S udy o P o ein-
nucleic acid
Complexes
Luis Filipe de Cas o Fe nandes
Mes ado em Química
Depa amen o de Química e Bioquímica
2013
O ien ado a
Ma ia João Ramos, P o esso a ca ed á ica, Faculdade de
Ciências da Uni e sidade do Po o
Co-o ien ado a
I ina Sousa Mo ei a, In es igado a ex e na, Faculdade de
Ciências da Uni e sidade do Po o
2
FCUP
S udy o P o ein-nucleic acid Complexes
FCUP
S udy o P o ein-nucleic acid Complexes
3
Todas as co eções de e minadas
pelo jú i, e só essas, o am e e uadas.
O P esiden e do Jú i,
Po o, ______/______/_________
4
FCUP
S udy o P o ein-nucleic acid Complexes
Acknowledges
O meu p imei o ag adecimen o e á que i pa a as minhas o ien ado as, em
especial pa a a p o esso a João, que me deu a opo unidade de abalha no g upo
mesmo es ando o a do país e de é ias quando a abo dei. Só mesmo es a
opo unidade pa a compensa a alha do es ágio na GALP. Depois que ia ag adece
odo o apoio e ajuda dos meus colegas de sala; O Rui, o João Ma ins e a Elisabe e
pelo ambien e descon aído e pela ajuda c ucial que me pe mi iu a ança ; os colegas
da sala do lado; o Zé, o Edua do, a Síl ia, o Rui e o João Ribei o, que es a am
semp e disponí eis pa a ajuda independen emen e da quan idade de abalho que
inham, sem eles não e ia conseguido ap ende an o; os colegas do ou o lado, o
Ósca , a Na é cia e o João Coimb a, que ambém es a am semp e lá! Semp e
dispos os a pa ilha o que sabiam pa a eu pode a ança ; e ambém o Sé gio e o
Nuno que com o seu conhecimen o eó ico me pe mi i am pe cebe onde es a a a
e a . Sem a ajuda de odos com ce eza não e ia ap endido an o e com ce eza não
e ia conseguido mon a es a in es igação e a sua consequência, es a ese. Pa a
acaba não posso deixa passa as qualidades des e g upo/ amília, conseguem passa
o espí i o de en e ajuda e o o gulho de pe ence ao g upo com a maio das
na u alidades. Não admi a que con inuem a c esce des a o ma!
Gos a a ambém de ag adece ao núcleo du o compos o pela Filipe, a Cláudia,
a Ana e a Tânia que o am o pila do p imei o ano de mes ado e o supo e do ano de
ese, espe o que es a amizade con inue pa a a ida.
Um ag adecimen o ambém ao Jumbo po me ajuda a paga as p opinas
desde a licencia u a.
E o g ande ob igado à Ca olina que em sido a minha aleg ia de i e nos
úl imos 8 anos, po odo o supo e, mo i ação e capacidade pa a me ob iga a le an a
nos momen os di íceis e a man e -me lá em cima quando es ou mo i ado. Sem i não
inha acabado a ese.
FCUP
S udy o P o ein-nucleic acid Complexes
5
Abs ac
The s udy o biological sys ems, hei s uc u e and unc ion is o g ea
impo ance o science and can lead o ad ances in he scien i ic knowledge and
con ibu ions o possible disco e ies om a pha maceu ical poin o iew. In his wo k
we s udied 4 P o ein-DNA complexes and 2 P o ein-RNA complexes using se e al
compu a ional echniques such as Molecula Dynamics simula ions (MD) and he
applica ion o he Alanine Scanning Mu agenesis (ASM) me hodology o he
iden i ica ion o ho -spo s (HS) and null-spo s (NS). We also made Roo Mean Squa e
De ia ion (RMSD) p o iles as well as Radial Dis ibu ion Func ion (RDF) and sol en
accessible su ace a ea (SASA) analysis.
The MD simula ions we e ca ied ou o 10ns in explici sol en , using he
99SB o ce ield o DNA-based complexes and na 99 o ce ield o RNA-based
complexes. A o al o 30 esidues om he 6 complexes (23 om DNA and 7 om
RNA), we e mu a ed o alanine and hei binding ee ene gy calcula ed and compa ed
o expe imen al alues. In he end we we e able o ge a good co ela ion wi h he
expe imen al alues wi h an a e age e o o 3.08 kcal/mol. These alues a e as
aluable as hey can be because he e a e e y ew s udies in his ield o DNA-based
complexes and no-one o RNA-based complexes. The e o e, i lea es a good base o
u u e s udies, u u e op imiza ions and u u e gene aliza ion and implemen a ion o his
kind o s udies.
We also complemen ed his s udy wi h RDF and SASA analysis o suppo O-
ing heo y ha s a es ha HS would be su ounded by egions wi h highe packing
densi y, mo e deeply bu ied. This leads o sol en exclusion a ound hem and esul s in
a lowe local dielec ic cons an en i onmen and enhancemen o speci ic elec os a ic
and hyd ogen bond in e ac ions. This egion would be su ounded by ano he one
o med by NS, whose ole would be o shel e he HS om bulk sol en .

6
FCUP
S udy o P o ein-nucleic acid Complexes
Keywo ds
Molecula Dynamics (MD), Alanine Scanning Mu agenesis (ASM), Radial Dis ibu ion
Func ion (RDF), Sol en Accessible Sol en A ea (SASA), P o ein-DNA, P o ein-RNA,
Ho -Spo s (HS), Null-Spo s (NS).
FCUP
S udy o P o ein-nucleic acid Complexes
7
Index
Acknowledges............................................................................................................... 4
Abs ac ........................................................................................................................ 5
Keywo ds ...................................................................................................................... 6
Index ............................................................................................................................. 7
Table index ................................................................................................................... 9
Figu e index ................................................................................................................ 10
Abb e ia ion Index ...................................................................................................... 12
1 - In oduc ion ............................................................................................................ 14
1.1 - Con ex ............................................................................................................ 14
1.2 - Ho spo s and he O- ing heo y ...................................................................... 15
1.3 - P o ein – P o ein in e aces ............................................................................. 16
1.4 - P o ein – nucleic acid complexes .................................................................... 17
1.5 - P o ein-based complexes ................................................................................ 18
1.5.1 - Esche ichia Coli eplica ion e mina o p o ein .......................................... 19
1.5.2 - DNA and p o ein NHP6A .......................................................................... 19
1.5.3 - DNA subuni RPA70 and Human eplica ion p o ein ................................. 20
1.5.4 - DNA and in eg ase p o ein TN916 ............................................................ 20
1.5.5 - U1A mu an and RNA complex ................................................................. 21
1.5.6 - RNA binding domain o Human ox-1 in complex UGCAUGU ................... 22
1.6 - Me hodology ................................................................................................... 23
1.6.1 - Compu a ional chemis y/Biochemis y ..................................................... 23
1.7 - Molecula Mechanics and o ce ield ............................................................... 24
1.7.1 - Ene gy minimiza ion ................................................................................. 28
1.7.2 - Molecula dynamic .................................................................................... 28
1.7.3 – Ensembles ............................................................................................... 28
1.7.4 In eg a ion ime ........................................................................................... 29
1.7.5 – Pe iodic bounda y condi ions ................................................................... 30
8
FCUP
S udy o P o ein-nucleic acid Complexes
1.8 - MM- PBSA ...................................................................................................... 31
1.8.1 - Sol a ion ................................................................................................... 32
1.9 - Alanine Scanning Mu agenesis ....................................................................... 33
2 - Me hodology .......................................................................................................... 35
2.1 - Sys ems p epa a ion ....................................................................................... 35
2.2 - Molecula Dynamics ........................................................................................ 36
2.3 - Alanine scanning mu agenesis ........................................................................ 36
2.4 - Sys em analysis .............................................................................................. 38
2.4.1 – Roo Mean Squa e De ia ion ................................................................... 38
2.4.2 – Radial Dis ibu ion Func ion ..................................................................... 39
2.4.3 – Sol en Accessible Sol en A ea .............................................................. 39
3 - Resul s and Discussion.......................................................................................... 40
3.1 - RMSD ............................................................................................................. 40
3.2 - RDF ................................................................................................................ 42
3.2.1 HS ≥ 2.0 kcal/mol cu -o ............................................................................. 43
3.2.2 HS ≥ 1.0 kcal/mol cu -o ............................................................................. 45
3.3 - SASA .............................................................................................................. 46
3.4 - Mu agenesis in P o ein acid nucleic in e aces ................................................ 53
Conclusion .................................................................................................................. 62
Re e ences ................................................................................................................. 65
Annexes...................................................................................................................... 70
RDF ........................................................................................................................ 70
MM-PBSA ............................................................................................................... 73
Pape – Ex ending he applicabili y o he O- ing heo y o p o ein-DNA complexes .... 76
FCUP
S udy o P o ein-nucleic acid Complexes
9
Table index
Table 1 - Di e ences be ween DNA and RNA ............................................................ 14
Table 2 – Cha ac e is ics o ab-ini io me hodologies ................................................... 23
Table 3 – Cha ac e is ics o Semi-empi ic me hodologies........................................... 23
Table 4 – Cha ac e is ics o Densi y Func ional heo ies me hodologies ..................... 24
Table 5 – Cha ac e is ics o Molecula Mechanics me hodologies .............................. 24
Table 6 - Composi ion o he 6 sys ems subjec ed o MD simula ions ......................... 36
Table 7 - Desc ip ion o he 30 esidues ha cons i u e he da ase , e idencing he
espec i e sys em, PDB nume a ion, amino acid ype and expe imen ally ∆∆Gbinding. .. 38
Table 8 - A e age numbe o wa e molecules a ound NS and HS o each complex
and global conside ing HS ≥ 2.0 kcal/mol. .................................................................. 43
Table 9 - A e age numbe o wa e molecules a ound NS and HS o each complex
and global conside ing HS ≥ 1.0 kcal/mol. .................................................................. 45
Table 10 – Resul s o ∆SASA and elSASA o all esidues wi h known ∆∆Gbinding o
he s udied complexes. ............................................................................................... 48
Table 11 - MM-PBSA esul s o ɛ1 o ɛ4 .................................................................... 54
Table 12 - MM-PBSA esul s o ɛ5 o ɛ9 .................................................................... 55
Table 13 - Resul s o a e age e o s ob ained wi h he NLPB equa ion. ..................... 56
Table 14 - Resul s o S a is ical es s o he 2.0 kcal/mol cu -o ................................ 57
Table 15 - Resul s o S a is ical es s o he 1.0 kcal/mol cu -o ................................ 59
16
FCUP
S udy o P o ein-nucleic acid Complexes
p o ein-RNA in e aces, ha e as much biological in e es as he p o ein-p o ein.
Howe e , he in o ma ion ega ding expe imen ally de ec ed HS in hese complexes o
he applica ion o he alanine scanning mu agenesis me hod o his ype o in e ace is
s ill sca ce. I p obably occu s due o he di icul ies in ene ge ic cha ac e izing his ype
o sys em as i possesses a highly cha ged cha ac e . Rega dless, i was obse ed he
same o ganiza ion o HS in he cen al egion o he in e ace bu wi h a di e en
composi ion. Fo p o ein-DNA in e aces he e is a highe occu ence o posi i ely
cha ged esidues (A ginine and Lysine), as well as, a lowe occu ence o hyd ophobic
and nega i ely cha ged esidues [22]. The iden i ica ion o HS can be made in
labo a o y (in i o o in i o). Among hem a e Ch oma in immunop ecipi a ion (ChIP),
whe e li ing cellules a e ea ed wi h o maldehyde o s abilize P o ein-DNA
in e ac ions allowing hei pu i ica ion and de ec ion, DNA elec opho e ic mobili y shi
essays, used o es he a ini y and speci ici y le els o he in e ac ions and mic opla e
cap u e and de ec ion assay, jus o name a ew. The compu a ional echniques o he
s udy o he ee ene gy di e ences upon alanine mu a ion o acid nucleic sys ems a e
no ully unde s ood. The e o e, we will s udy how o implemen in silico de ec ion o
HS in hese sys ems as well as hei cha ac e is ic accessibili y o sol en .
1.3 - P o ein – P o ein in e aces
P o ein - p o ein in e ac ions (PPIs) a e in ol ed in a wide a ie y o cellula
p ocesses and a e c i ical e en s in mos biological pa hways and hei unc ion o
mal unc ion esul s in a a ie y o diseases, u ning hese in e aces compelling a ge s
o d ug disco e y [23, 24] . The e ha e been se e al a emp s o unde s and hem in
e ms o physical ea u es o he associa ing su aces and ene ge ic con ibu ions made
by each esidue [25]. This iden i ica ion o he key esidues ha a e impo an o he
in e ac ion is e y di icul , due in pa o an incomple e unde s anding o he sou ces o
a ini y and speci ici y o in e aces [24]. An accu a e unde s anding o he ac o s ha
make ce ain esidues mo e impo an han o he s in acili a ing hese in e ac ions is
also going o be o eno mous impo ance a edesigning he a ini y and speci ici y o
na u al occu ing in e ac ions as well as o new p o ein-p o ein designs [25]. The MM-
PBSA (molecula mechanics-Poisson Bol zmann su ace a ea) is widely used o
in es iga e PPIs and o he in e ac ions o a ound a decade combining he speed o a
con inuum app oach o modelling sol en in e ac ions wi h heo e ical accu acy o an
MM-based app oach o a omis ically modelling p o ein-p o ein in e ac ions. In o de o

FCUP
S udy o P o ein-nucleic acid Complexes
17
p edic he loca ion o HS a in e aces, hey also ha e being used in a ious alanine-
scanning mu agenesis p o ocols, calcula ing he ela i e ee ene gy change (∆∆Gbinding)
be ween he wild- ype and mu an complex upon alanine mu a ion.[23, 26].
1.4 - P o ein – nucleic acid complexes
P o ein-DNA in e ac ions play an essen ial ole in many cellula unc ions such
as ansc ip ion, eplica ion, ecombina ion and DNA packaging. P o ein-RNA
in e ac ions a e also essen ial in biological p ocess and some o hei unc ions a e
ansc ip ion e mina ion, mRNA splicing, mRNA expo o he nucleus and cy oplasm,
in acellula localiza ion o ansc ip s, mRNA ansla ion, mRNA s abili y and
p ocessing RNA and RNA [27]. Since he de elopmen o compu a ional me hods
powe ul and eliable enough he e has been a con inuous g ow h in he numbe s o
P o ein-DNA s uc u es a ailable o s udy. (P o ein Da a Bank[28] - PDB) and hey a e
c ea ed using X- ay c ys allog aphy.
All he esea ch wo k done on his ma e elies on he quali y ( esolu ion) o he
s uc u es. Wi h a highe esolu ion, be e esul s a e possible ega ding in e ac ion
be ween a oms and be ween molecules, o ganiza ion and eo ganiza ion o base
sequences and s uc u e modi ica ion a e bond b eaking/ o ma ion. The e a e o he
use ul da abases ha can comple e PDB’s in o ma ion like P oNIT(The modynamic
da abase o p o ein-nucleic acid in e ac ions) [29-31] whe e can be ound expe imen al
da a o se e al he modynamic and ene gy pa ame e s o P o ein-DNA and P o ein-
RNA complexes and AANT (amino acid-nucleo ide in e ac ion da abase) [32] whe e
can be ound s a is ical in o ma ion ega ding aminoacid-nucleo id in e ac ions.
DNA s uc u es can be classi ied acco ding o hei unc ion and can be di ided
in h ee classes: (i) Enzyme – i i s p ima y unc ion is he modi ica ion o DNA; (ii)
T ansc ip ion ac o – i i s unc ion id he egula ion o he exp ession and ansc ip ion
o genes (iii) Suppo p o ein – i i s unc ion is solely o p o ide DNA suppo . These
classes can be u he di ided in o ypes conside ing hei unc ion and s uc u e: (a) o
enzymes we ha e 6 sub-ca ego ies ( oxido educ ases, ans e ases, hyd olases,
lyases, isome ases and ligases); (b) o ansc ip ion ac o s we ha e 7 sub-ca ego ies
( Alpha Helix, Alpha/Be a, Be a Shee , Helix u n Helix, Ribbon/Helix/Helix, Zinc
Coo dina ing and Zippe ype); (c) o suppo ing P o eins we ha e 8 sub-ca ego ies[33,
34].
18
FCUP
S udy o P o ein-nucleic acid Complexes
RNA s uc u es can be classi ied acco ding o hei unc ion and can be di ided
in wo majo classes: (i) ibosomal RNA ha ensu es he co ec p o ein sequence
co ec ing missing codons and (ii) RNA polyme ase ha ecognizes he co ec
sequence and syn he izes i .
1.5 - P o ein-based complexes
In he ollowing sub sec ions we will gi e a e y ligh desc ip ion o each
complex unde s udy.
Figu e 1 - Rep esen a ion o he 4 p o ein-DNA complexes and 2 p o ein-RNA complexes s udied in his wo k. P o ein
and DNA a e in ca oon and s ick ep esen a ion espec i ely
1ECR
1J5N
1JMC
1TN9
1URN
2ERR
FCUP
S udy o P o ein-nucleic acid Complexes
19
1.5.1 - Esche ichia Coli eplica ion e mina o p o ein
The Esche ichia Coli eplica ion e mina o p o ein (PDBid: 1ECR [35]), occu s
a disc e e Te si es. These si es block eplica ion o k p og ession in i o, when he
eplica ion o k app oaches om on di ec ion, he non-pe missi e di ec ion, he e is a
unc ion c ea ing a ap ha es ic s he mee ing o he con e gen o ks o a ce ain
ch omosome o a ce ain egion.[36] The eplica ion o DNA in many p oka yo es and in
ce ain egions o euka yo ic ch omosomes is speci ically e mina ed a specialized
sequences called eplica ion e mini, Te , ha cause o ien a ion-dependen o ks a es ,
which pe o ms impo an physiological unc ions[37]. The DNA eplica ion e mina ion
p o ein, TUS, blocks he p og ess o he eplisome in he inal s ages o he
ch omosomal eplica ion in Esche ichia Coli and ela ed bac e ial species [38]. In i o
analyses ha e shown ha he eplica ion e mina o p o ein o Esche ichia Coli is a
pola con ahelicase, meaning, he p o ein causes a unidi ec ional a es o he
eplica i e helicase DnaB upon binding o he e sequence.[37] The c ys al s uc u e o
he Tus-Te complex indica es ha he co e DNA-binding domain o he p o ein
consis ing in wo pai s o an ipa allel be a-s ands ha lie in he majo g oo e o he
DNA. [38]
1.5.2 - DNA and p o ein NHP6A
The complex be ween DNA and he p o ein NHP6A (PDBid: 1J5N [39]), is a
HMG box p o ein ha can be ound in Saccha omyces ce e isiae. HMG is he ac onym
o High mobili y g oup, and i ’s a conse ed domain o ~80 amino acids wi ch media es
DNA binding o many p o eins.[40]The i s class (HMG1) is gene ally ansc ip ion
ac o s ha bind o DNA in a sequence speci ic ashion and a e exp essed only in a ew
cell ypes, con aining only one HMG box, while he second class(HMG2) is mo e
abundan and o en con ains wo o mo e HMG boxes, binding o DNA wi h li le o no
sequence speci ici y[40]. HMG p o eins a e small ch oma in associa ed euka yo ic
p o eins ha al e he physical p ope ies o DNA in i o and in i o[41]. These p o eins
a e membe s o a class o small p o eins ha a e abundan in euka yo ic cells and a e
sequence-nospeci ically bind o DNA.[42] The e a e wo g oups o HMGs, he A g oup
and he B g oup and hey di e in e ms o shape and o ien a ion o i s i s al a-helix
and he iden i y o po en ial in e cala ing esidues. The A box domains is known o bend
20
FCUP
S udy o P o ein-nucleic acid Complexes
less DNA[43]. Each homologous mo i con ains amino acids ha o m h ee al a helices
o bind DNA as an L-shaped s uc u e. [41]
1.5.3 - DNA subuni RPA70 and Human eplica ion p o ein
The complex (PDBid: 1JMC [44]) is he ep esen a ion o he human eplica ion
p o ein (RPA) which is a key ac o in DNA me abolism including DNA eplica ion, DNA
epai and ecombina ion [45]. I ’s a modula mul i domain p o ein ha unc ions in a
wide ange o DNA pa hways equi ed o main ain and p opaga e he genome o all
li ing o ganisms[46], i s cons i u ed by a s able single s anded DNA binding p o ein
composed by h ee subuni s (70kDa, 32kDa and 14kDa; RPA70, RPA32 and RPA14
espec i ely)[45, 47, 48]. RPA unc ion by in e acing wi h dynamic mul i-p o ein
machine y and ac s as a cen al hub ha links many DNA ansac ions, i also p o ides
he p ima y single-s anded DNA (ssDNA) binding ac i i y in euka yo es and e en
se e s as a sca old and coo dina o o DNA p ocessing machine y[46]. RPA is highly
conse ed h oughou e olu ion, and homologous, he e o ime ic single s anded DNA-
binding p o eins ha e being iden i ied in all euka yo es examined [45]. The p ima y
in e ac ion o RPA is wi h ssDNA, howe e , RPA unc ion equi es in e ac ions wi h
o he o ms o DNA, i binds o damaged DNA and double s anded DNA (dsDNA) and
can cause dsDNA helices des abiliza ion, his des abiliza ion is a mani es a ion o
ssDNA ac i i y[47].
1.5.4 - DNA and in eg ase p o ein TN916
The c ys al s uc u e o he DNA binding domain o Tn916 in eg ase (PDBid:
1TN9 [49]) is essen ial o excision and ein eg a ion o bac e ial Tn916 conjuga i e
ansposon and he la e sp eads an ibio ic esis ance among pa hogenic bac e ia[50].
Tn916 is a conjuga i e ansposon (also called In eg a i e conjuga i e elemen s, ICEs
[51]), and like mos ansposons is ex emely p omiscuous gene ic elemen ha
dissemina es an ibio ic esis ance among g am posi i e and g am nega i e
bac e ia[52], se ing as a majo con ibu o o bac e ial e olu ion by passing he
an ibio ic esis ance, i ulence genes and me abolic genes ac oss species and genus
lines [51]. Tn916 is also o he mos ex ensi ely s udied ansposon.
FCUP
S udy o P o ein-nucleic acid Complexes
21
Unlike mos DNA-binding domains epo ed ha bind o majo g oo e using -
helix [53], he Tn916 N- e minal domain (INT-DBD) ecognizes he majo g oo e using
he ace o a h ee-s anded be a-shee . The majo p o ein-DNA con ac s occu a he
la gely hyd ophobic in e ace o med by u n T1 and s ands Be a2 and be a3[50]. This
N- e minal domain, INT-DBD ecognizes DNA by a a e s uc u al mo i , he h ee
s anded be a shee [54].
1.5.5 - U1A mu an and RNA complex
The c ys al s uc u e o an RNA ecogni ion mo i (RRM) is also known as
ibonucleop o ein (RNP) consensus domain o RNA binding domain (RBD). I is
cha ac e ized by highly conse ed egions loca ed cen ally on a be a shee , which
o ms he RNA binding su ace [55-57], his domain is he hi d mos common in human
p o eins [58] (PDBid: 1URN [59]). I is p esen in one o mo e copies in hund eds o
RNA binding domains and p o eins ha ca y RRM domains play c i ical oles in a wide
a ie y o cellula p ocesses, including RNA p ocessing and packaging, mRNA expo ,
ansla ion, RNA deg ada ion and gene egula ion [27, 55, 56, 58].
These domains a e abou 90 amino acids long and old in o a globula s uc u e
consis ing o a ou -s anded an ipa allel be a-shee ( he RNA binding su ace) backed
by wo al a-helices and a e cha ac e ized by he p esence o wo highly conse ed
s e ches o 8 and 6 amino acids, known as RNP1 and RNP2 consensus sequences,
which lie s a egically in he cen e o he be a shee su ace and domain conse ed
a oma ic esidues c i ical o RNA binding [55, 56, 58], con as ing o mos DNA-binding
p o eins, which a e p esen ed wi h a double-s anded b- o m helix o uni o m s uc u e.
RNA-binding s uc u es mus be able o bind a ge s wi h widely di e ing s uc u es and
mus be able o bind o i s co ec RNA a ge wi h app op ia e kine ics, a ini ies ha
co espond o he unc ion o he complex, anging om ela i ely nonspeci ic,
ansien binding (such as he binding in ol ed in gene al RNA p ocessing), o highly
speci ic and s able in e ac ions (such as hose in ol ed in he o ma ion o in acellula
machine y) [27, 56].This ecogni ion is done by bo h sequence and s uc u e displaying
a conside able a ie y in he binding a ini ies [57]. Because he s eep and na ow
g oo e o double s anded RNA does no p o ide p o eins easy access o he bases o
sequence-speci ic ecogni ion, mos RNA-binding p o ein ecognize single-s anded
egions o dis o double-s anded egions in which he majo g oo e has been widened
by bulges, hai pins o loops [56].

22
FCUP
S udy o P o ein-nucleic acid Complexes
1.5.6 - RNA binding domain o Human ox-1 in complex UGCAUGU
The RNA elemen UGCAUGU ( ep esen ed wi h PDBID:2ERR [60]) has long
known o s ongly in luence splicing o a a ie y o al e na i e exons in mammalian
genes, including he c-s c N1 exon, he calci onin/CGRP exon4, he ib onec in exon
IIIB, he ib oblas g ow h ac o ecep o 2 exon and he nonmuscle myosin II hea y
chain B exon N30 [60, 61]. RNA splicing plays a c i ical ole in he p og amming o
neu onal di e en ia ion and has a consequence in human neu ode elopmen [62] so
genes a ge ed by neu onal FOX-1 a e much mo e likely o be in ol ed in neu onal
cy oskele al ea angemen s and neu onal esicula and p o ein anspo unc ions, as
an example, analysis o RNA ecogni ion si es cha ac e ized o b ain speci ic Fox-1
showed ha hese sequences a e highly ep esen ed in al e na i ely spliced ansc ip s
p e e en ially exp essed in neu ons [63].
The ox-1 gen was o iginally iden i ied in Caeno habdi is elegans, whe e i ac s
as a nume a o elemen in coun ing he numbe o X ch omosomes ela i e o ploidi y,
and de e mining male o he maph odi e de elopmen . I is hough o pos -
ansc ip ionally ep ess he exp ession o Xol-1 ( he main swi ch con olling sex
de e mina ion). Bu since se e al al e na i ely spliced iso o ms o Xol-1 exis while only
one o hese splice a ian s is necessa y and su icien as a sex de e minan , i was
specula ed ha Fox-1 migh led o unp oduc i e splicing o he Xol-1 gene [60, 61]. The
Fox-1 amily o RNA binding p o eins a e egula ed by al e na i e splicing in neu ons,
so Fox-1 and alike p o eins a e exp essed p edominan ly in b ain, skele al muscle and
ca diac muscle [63, 64]. The Fox-1, in addi ion o he nume ous hyd ophobic and
elec os a ic in e ac ions ha p o ide a ini y, also has a dense ne wo k o hyd ogen
bonds ha p o ide sequence speci ici y o he i s six nucleo ides 5’-UGCAU-3’, being
he mos impo an in e ac ions a he p o ein-RNA in e ace [60].
FCUP
S udy o P o ein-nucleic acid Complexes
23
1.6 - Me hodology
1.6.1 - Compu a ional chemis y/Biochemis y
Since he exponen ial de elopmen o compu e s o e he las decades, i was
possible o me ge he adi ional expe imen al Chemis y/Biochemis y wi h he
p ocessing abili y o machines gi ing bi h o a new way o c ea e science. I was he
c ea ion o Compu a ional Chemis y and Compu a ional Biochemis y. This new way o
doing science allowed scien is s among o he hings o s udy and simula e a g ea deal
o p o ein-based sys ems and o desc ibe i s p ope ies such as molecula s uc u e,
in e ac ions be ween i s componen s, geome y and a as a ay o he modynamic
p ope ies. One o he main goals has been he c ea ion and disco e y o new d ugs
h ough hi /lead me hodologies and hei op imiza ion. The a ious me hodologies used
can be spli in ou majo g oups wi h i s ad an ages, disad an ages and speci ic
a ge s as shown in he Tables 2, 3, 4 and 5.
ab-ini io
Desc ip ion
- Uses quan ic physics
- Does no include empi ic pa ame e s
- Ma hema ical igo ous
- Based on wa e unc ions
Ad an ages
- Can be used in all kind o sys ems
- Does no depend on expe imen al da a
- Allows calcula ion o ansi ion and exci ed s a es
Disad an ages
- Ve y demanding om he compu a ional iew
Sys ems s udied
- No o e a ew hund ed a oms
Table 2 – Cha ac e is ics o ab-ini io me hodologies
Semi-empi ic
Desc ip ion
- Uses quan ic physics
- Uses expe imen al pa ame e s and empi ic
simpli ica ions
- Includes se e al app oxima ions
- Based on wa e unc ions
Ad an ages
- Less demanding om he compu a ional iew when
compa ed wi h ab-ini io and DFT me hods
- Allows calcula ions o ansi ion and exci ed s a es
Disad an ages
- Requi e expe imen al da a o ab-ini io calcula ions o he
pa ame e de i a ion
- Less igo ous han he ab-ini io me hods
Sys ems s udied
- No abo e he housands o a oms
Table 3 – Cha ac e is ics o Semi-empi ic me hodologies
24
FCUP
S udy o P o ein-nucleic acid Complexes
DFT (Densi y Func ional Theo y)
Desc ip ion
- Based on densi y unc ionals
- Uses pa ame e s on he unc ionals
Ad an ages
- Include he elec onic co ela ion e m
- Less demanding om he compu a ional iew han ab-
ini io me hods, using he same quali y o calcula ions
- The esul s achie ed a e be e in sys ems o open laye
- Allows calcula ion o ansi ion s a es
Disad an ages
- Less igo ous han he ab-ini io me hods when he
unc ionals don’ adap o he pa ame e s o calcula e
- No possible o calcula e exci ed s a es
- No possible o imp o e sys ema ically he esul s
- No e y accu a e o desc ibe dispe si e in e ac ions
Sys ems s udied
- No abo e he hund ed a oms
Table 4 – Cha ac e is ics o Densi y Func ional heo ies me hodologies
Molecula mechanics
Desc ip ion
- Based on he laws o he classic mechanics
- Use o o ce ields based on empi ical pa ame e s
Ad an ages
- The calcula ions a e e y as and use ul specially when
he compu a ional esou ces a e sca ce and he
pa ame e s used a e adequa e o he sys em
- Can be used o s udy la ge sys ems like enzymes
Disad an ages
- Does no calcula e elec onic p ope ies
- Requi e expe imen al da a o ab-ini io calcula ions o he
de i a ion o pa ame e s
- Fo ce ield use is limi ed o a ce ain ype o sys em
Sys ems s udied
- Sys ems may ha e hund eds o housand a oms
Table 5 – Cha ac e is ics o Molecula Mechanics me hodologies
The me hod used mus be adequa e o he sys em s udied and in some cases
mo e han one me hod can be used. One example o ha is using a hyb id me hod like
QM/MM (Quan um Mechanics/Molecula Mechanics), in which we can ha e he
ad an ages o he quan um physics like he p ecision o he quan ic physics and he
speed o he molecula mechanics in he s udy o chemical p ocesses in solu ion o in
p o eins. The quan um mechanics is used o s udy he smalle pa s o he sys em like
he nucleus and molecula mechanics o s udy he es o he sys em.
1.7 - Molecula Mechanics and o ce ield
This is he mos sui able me hod o s udy p o ein sys ems, since i equi es a
lowe le el o compu a ional powe han quan ic me hods. Like said in Table 5, i uses
he laws o classic mechanics and New on Laws o desc ibe he pa icles mo emen .
FCUP
S udy o P o ein-nucleic acid Complexes
25
Molecula mechanics calcula ions, also known as o ce ield calcula ions, can
be o conside able use in he quali a i e desc ip ions o sys ems. In hese cases, we
concen a e on he s uc u al aspec and no on he elec onic and/o spec oscopic
p ope ies. In essence, we desc ibe he po en ial ene gy su ace wi hou in oking any
quan um mechanical calcula ions o desc ip ions. The Bo n-Oppenheime
app oxima ion, undamen al o ou molecula desc ip ion, s a es ha he Sch odinge
Equa ion o a molecule can be sepa a ed in o a pa desc ibing he mo ions o he
elec ons and a pa desc ibing he mo ions o he nuclei and ha hese wo mo ions
can be s udied independen ly. This can be in e p e ed in one o wo manne s, one o
which allows he s udy o he elec onic s uc u e, one o which allows he s udy o he
molecula mechanics s uc u e. Bu sine his me hod only conside s he mos impo an
nuclea mo emen s o desc ibe he molecule and does no conside i s elec ons, i is
no capable o acknowledge he o ma ion and b eak o bonds and elec onic exci ed
s a es.
In molecula mechanics, he smalles pa icle o he sys em is he a om, so he
nuclei and he elec ons a e ea ed using pa ame e iza ion wi h a o ce ield. In
molecula mechanics a o ce ield is a ma hema ic exp ession o physical a iables o
desc ibe he po en ial ene gy o a sys em. The e is a common exp ession o all o ce
ields o calcula e he ene gy:
The bond s e ching (ene gy equi ed o s e ch o comp ess a bond be ween
wo a oms), bending (ene gy equi ed o bend a bond om i s equilib ium angle) and
o sional ( o sional ene gy o dihed al angles) e ms a e called bonded in e ac ions
because he a oms in ol es mus be di ec ly bonded o bonded o a common a om.
The Van de Waals (ene gy esponsible o he lique ac ion o non-pola gases like O2
and N2, also go e n he ene gy o in e ac ion o non-bonded a oms wi hin a molecule.
These in e ac ions con ibu e o he s e ic in e ac ions in molecules and a e o en he
mos impo an ac o s in de e mining he o e all molecula con o ma ion (shape),
being he mos impo an o de e mine he h ee dimensional s uc u e o many
biomolecules, especially p o eins) and elec os a ic ( when bonds in he molecule a e
pola , pa ial elec os a ic cha ges will eside on he a oms. These in e ac ions a e
ep esen ed wi h a Columbic po en ial unc ion) e ms a e be ween non-bonded a oms.
The las e m co ela e he p e ious ones, bu is o en omi ed because i inc eases
g ea ly he compu a ional ime.
32
FCUP
S udy o P o ein-nucleic acid Complexes
1.8.1 - Sol a ion
The ee ene gy o each esidue was es ima ed as he sum o he molecula
mechanical ee ene gy, he sol a ion ee ene gy and he con ibu ions om he
ib a ional, o a ional and ansla ion en opy. This ene gy can be di ided in o pola
(Gpola ) and nonpola (Gnp) con ibu ions (equa ion 12).
[12]
The nonpola sol a ion e m includes he ene ge ic cos o he ca i y o ma ion,
sol en e-a angemen and in e ac ions be ween sol en -solu e, so, his e m
ep esen s he ee binding ene gy o he molecule when i s emo ed om all cha ged
(pa ial cha ges a e aken as ze o) as seen in equa ion 13).
∆Gdispe sion is he ene gy o he Van de Waals in e ac ions be ween sol en -
solu e and he ∆Gca i y e m includes he en opic penaliza ion due o he e-
a angemen o he sol en molecules a ound he solu e and he wo k ealized o c ea e
he ca i y needed o make he solu e eme ge. Bo h e ms a e p opo ional o solu es
SASA making he nonpola e m able o be es ima ed wi h equa ion 14, Whe e A is he
SASA alue es ima ed by Molsu so wa e included in he AMBER package and σ and
β a e empi ic cons an s wi h alues o 0.00542 kcal Å-2 mol-1 and 0.92 kcal mol-1
espec i ely.
The pola sol a ion e m can be calcula ed by sol ing he Gene alized Bo n
(GB)[82, 83] equa ion o by he app oxima ion Poisson Bol zmann (PB)[84]. Since he
PB model is conside ed he mos p ecise i was used as e e ence in GB models which
a e conside ed o be mo e e icien in a compu a ional poin o iew. Since his me hod
was designed o P o ein-P o ein in e aces, i s esul s on P o ein-Nucleic acid a e no
as accu a e, and o sol e his p oblem, i was needed o use a di e en me hod o
calcula e he pola sol a ion e m. The p og am used was DelPhi [85, 86] which uses a
di e en app oach whe e he p o ein is modelled as a dielec ic con inuum o low
pola izabili y embedded in a dielec ic medium o high pola izabili y[87]. The Gpola sol a ion
was calcula ed by sol ing he Linea Poisson−Bol zmann (LPB) equa ion, he
adi ional me hod, and he Nonlinea Poisson−Bol zmann (NLPB) equa ion, which
accoun s o he impo ance o sal concen a ion in he medium. This ac o is

FCUP
S udy o P o ein-nucleic acid Complexes
33
pa icula ly impo an in p o ein−DNA in e aces due hei highly cha ged and pola
cha ac e . Wi h his in mind, we used a alue o 2.5 g ids/Å o scale; a alue o 0.001
kT/c o he con e gence c i e ion; a 90% o he ill o he g id box; and he Coulombic
me hod o se he po en ials a he bounda ies o he ini e-di e ence g id. The dielec ic
bounda y was aken as he molecula su ace de ined by a 1.4 Å p obe sphe e and by
sphe es cen e ed on each a om wi h adii aken om he Pa se33 dW adii pa ame e
se . The sal concen a ions used we e 0.010 M and 0.145 M, which a e in he
physiological ange.34 We ha e also calcula ed he elec os a ic sol a ion ene gy e m
using PB sol e implemen ed in he pbsa module om he AMBER package. We es ed
a se o nine di e en dielec ic cons an s, om 1 o 9, o mimic he expec ed
ea angemen upon alanine mu a ion and o assess he impo ance o each dielec ic
cons an in he de e mina ion o ∆∆Gbinding.
1.9 - Alanine Scanning Mu agenesis
Alanine-Scanning Mu agenesis is an ex ension o he MM-PBSA and can be
used o iden i y mu a ions ha can enhance he binding a ini ies o he complex due o
i s abili y o es ima e he con ibu ion o each esidue o he p o ein-p o ein, p o ein-
DNA/RNA o p o ein-ligand binding[78]. I ’s also one o he mos used me hods o
analyse and de ec HS and o s udy he unc ional g oups o he la e al chains o he
amino acids in speci ic poin s. I wo ks by eplacing he o iginal esidue wi h an alanine
and calcula ing i s ee binding ene gy o compa e wi h he o iginal esidue’s ee
binding ene gy.
In his wo k we e conside ed wo ways o ecognizing HS since he e s ill isn’ a
consensual alue. So, i s HS we e conside ed o ha e a ee binding ene gy o 1.0
kcal/mol upon alanine mu a ion and hen HS we e conside ed o ha e a ee binding
ene gy o 2.0 kcal/mol.
34
FCUP
S udy o P o ein-nucleic acid Complexes
Figu e 3 - Schema ic ep esen a ion o he ini ial me hod o mula ion o he de e mina ion o ∆∆Gbinding.
Wild- ype s uc u e
MD simula ion wi h explici
sol en ep esen a ion
ASM calcula ion and
analysis using elec os a ic
and pola sol a ion e ms
ɛ1 o ɛ9
∆∆Gbinding
FCUP
S udy o P o ein-nucleic acid Complexes
35
2 - Me hodology
2.1 - Sys ems p epa a ion
The i s s ep was o ind P o ein-DNA sys ems wi h known expe imen al da a
o ee binding ene gies upon alanine mu a ion o he in e acial esidues. To his end
we used he P oNIT da abase. [28-30] Six di e en complexes, 4 P o ein-DNA and 2
P o ein-RNA we e s udied (Figu e 1) o a o al o 30 esidues: (i)The p o ein -
eplica ion- e mina ion-p o ein and DNA (PDBid: 1ECR[35]); (ii) he nonhis one
ch omosomal p o ein and DNA (PDBid: 1J5N [39]) (iii) he human eplica ion p o ein A
and DNA (PDBid: 1JMC [44]); (i ) he N- e minal domain o he Tn916 in eg ase p o ein
bound o i s DNA-binding si e (PDBid: 1TN9 [49]); ( ) he p o ein U1A and RNA
(PDBid:1URN[49]) and ( i) A axin-2-binding p o ein 1 and RNA (PDBid: 2ERR[60]).
Then, we e ie ed he 3D s uc u es om he PDB [28] and p ocess hem. We
i s p o ona e he amino acids since he c ys allog aphic s uc u es in PDB do no
possess enough esolu ion o ha e he hyd ogen a oms. To access he p o ona ion
s a e o esidues we used he P opka [88-90] so wa e wi hin he PDB2PQR [91, 92]
se e .
Then he leap p og am included in he AMBER[69] package was used o c ea e
he necessa y inpu iles o un he MD simula ions using he 99SB o ce ield o
p o ein-DNA complexes and na 99 o p o ein-RNA complexes. Leap was used o
sol a e each sys em wi h a 10Å TIP3P [93, 94] wa e box. An app op ia e numbe o
Na+ ions we e added o p ope ly neu alize he sys em and he inpu iles o he
simula ion we e sa ed: he opology one (. op) and he coo dina es one (.c d). The
composi ion o he sys em in s udy is summa ized in Table 6.
36
FCUP
S udy o P o ein-nucleic acid Complexes
Complex
Residues
A oms
AA
DNA
Wa e s
Ions
To al
1ECR
305
30
13052
20Na+
13407
45164
1J5N
93
30
9791
22Na+
9936
31886
1JMC
238
8
11408
10 Na+
11664
38207
1TN9
69
26
7963
20Na+
8078
25905
1URN
96
21
7716
11Na+
7833
25377
2ERR
109
7
6388
3Na+
6504
20805
Table 6 - Composi ion o he 6 sys ems subjec ed o MD simula ions
2.2 - Molecula Dynamics
The MD simula ions we e execu ed in h ee s eps: (i) Minimiza ion s ep; (ii)
Hea ing un and (iii) P oduc ion un. The minimiza ion s ep is equi ed o elimina e bad
con ac s in he c ys allog aphic s uc u es and he in e ac ion o he p o eins wi h he
sol en . We used he SANDER module in AMBER09[69] package. The sys ems we e
subjec o 2 ns o hea ing whe e he empe a u e was g adually inc eased since 0 o
300K wi h an ensemble NVT, ollowed wi h 8 ns o p oduc ion wi h an ensemble NPT.
The Lange in algo i hm was used o egula e he empe a u e o he sys em. The
Pa icle Mesh Ewald (PME) was used o ea elec os a ic in e ac ions o long ange,
being he non-ligand in e ac ions blocked o a 10 Aº adius. In e e y simula ion, he
SHAKE[76] algo i hm was used o cons ain all co alen bonds in ol ing hyd ogen
a oms. The in eg a ion s ep was 2 s. Fo all sys ems we e execu ed 10 ns simula ions
using explici sol en and he 99SB o ce ield o DNA-based complexes and na 99
o ce ield o RNA-based complexes.
2.3 - Alanine scanning mu agenesis
MM-PBSA me hod was used o calcula e he bond ee ene gies a e alanine
mu a ion o he in e acial esidues. Equa ion 4 was used o calcula e his ee ene gy.
The en opic e m o his equa ion ( he las one), can be neglec ed since i s pa ial
con ibu ions end o be negligible. The i s h ee e ms we e in oduced he way hey
FCUP
S udy o P o ein-nucleic acid Complexes
37
a e gi en by he me hod, bu he ee ene gy o pola sol a ion had o be calcula ed
using he esolu ion o he linea and non-linea equa ion o Poisson-Bol zmann
ecu ing o DelPhi so wa e. In his con inuum me hod, he p o ein is modelled as a
dielec ic con inuum o low pola izabili y embedded in a dielec ic medium o high
pola izabili y. Because he ollowing pa ame e s ha e been shown in ea lie wo ks o
cons i u e a good comp omise be ween accu acy and compu ing ime, hey we e se
as: (i) a scale o 2.5 g ids/Aº; (ii) a con e gence c i e ion o 0.001 kT/c and (iii) he
molecule illed 90% o he g id box.
The dielec ic bounda y was aken as he molecula su ace de ined by a 1.4Aº
p obe sphe e and by sphe es cen ed in each a om. To e i y he mos co ec
dielec ic cons an ha should be used o simula e he ea angemen o he p o ein
upon alanine we used alues om 1 o 9. To ease he sys ema ic wo k equi ed, we
used a VMD plugin called CompASM [95] which p o ides an easy way o p epa e inpu
iles and analyse esul s h ough he use o a g aphical in e ace. The mu a ed esidues
a e lis ed in Table 7.
P o ein
#AA PDB
#AA Mu a ed
Gbinding / kcal mol-1
e e ences
1ECR
198
R
1.19
[38]
1ECR
250
Q
0.11
1J5N
22
K
0.43
[40]
1J5N
23
R
0.63
1J5N
28
Y
0.90
1J5N
29
M
0.46
1J5N
33
N
0.74
1J5N
36
R
0.84
1J5N
40
R
0.72
1J5N
48
F
0.40
1J5N
53
K
0.49
1J5N
54
K
0.00
1J5N
58
K
0.00
1J5N
60
K
0.40
1J5N
67
K
0.25

38
FCUP
S udy o P o ein-nucleic acid Complexes
1J5N
78
K
0.43
1J5N
81
Y
0.21
1J5N
88
Y
0.36
1JMC
238
F
0.20
[45]
1JMC
361
W
1.09
1JMC
234
R
2.15
[47]
1JMC
277
E
1.36
1JMC
382
R
1.91
1TN9
15
T
0.08
[53]
1URN
51
M
0.54
[55]
1URN
54
Q
4.85
[58]
1URN
56
F
3.23
2ERR
120
H
2.98
[60]
2ERR
126
F
4.31
2ERR
158
F
3.87
2ERR
160
F
6.08
Table 7 - Desc ip ion o he 30 esidues ha cons i u e he da ase , e idencing he espec i e sys em, PDB nume a ion,
amino acid ype and expe imen ally ∆∆Gbinding.
2.4 - Sys em analysis
2.4.1 – Roo Mean Squa e De ia ion
The i s s ep o he analysis was he de e mina ion o he RMSD (Roo Mean
Squa e De ia ion) which o in e he s abili y o he complexes and monome s along he
MD simula ion. To pe o m his calcula ion, i was used he PTRAJ p og am which is
included in AMBER9 [69] package.
FCUP
S udy o P o ein-nucleic acid Complexes
39
2.4.2 – Radial Dis ibu ion Func ion
The measu emen o he Radial Dis ibu ion Func ion allows he
cha ac e iza ion o he in e ac ion be ween he solu e and he sol en molecules. The
wa e molecules a ound he p o ein complexes can be di ided in h ee ca ego ies: (i)
Wa e molecules in ol ing he p o ein s uc u e, which a e ee o mo e hemsel es
and assis in he p o ein di usion compa ing o he o he molecules by mo ing casually
in he solu ion; (ii) hyd a ion wa e molecules on he p o ein su ace; and (iii) indi idual
wa e molecules connec ed o each o he and o ming hyd ogen b idges wi h cha ged
and pola esidues, s abilizing he p o ein s uc u e. Wi h his, is possible o ge he
densi y o he sol en pa icles ha a e a a dis ance om he solu e pa icles.
2.4.3 – Sol en Accessible Su ace A ea
SASA is he ac onym o Sol en accessible Su ace a ea and is a way o
quan i ying hyd ophobic bu ial, by o he s wo ds i desc ibes he a ea a ound he p o ein
on which is possible o occu in e ac ions wi h he sol en . The e o e, SASA is he
sol en -accessible su ace a ea ha was es ima ed using he MSMS algo i hm wi h
p obe adius o 1.4 Å. In house sc ip s we e used in he VMD o pe o m his
calcula ion.
Fo all esidues SASA calcula ions we e done wi h he objec i e o ge ing o
know he impo ance o wa e molecules a ound HS and NS. These calcula ions we e
done in he las 2ns o he MD in explici sol en . In each case we ha e calcula ed
SASA o he complex (SASAcpx) and he monome (SASAmon). ∆SASA and elSASA
we e also calcula ed as elSASA allows he di e en ia ion o esidues wi h equal
∆SASA bu di e en sol en exposu e; his was done acco ding o equa ions 15 and
16.
40
FCUP
S udy o P o ein-nucleic acid Complexes
3 - Resul s and Discussion
In his wo k we e s udied 30 mu a ions in 6 sys ems, s a is ically he di e en
amino acids can be dis ibu ed: A g (17%), Lys (24%), Glu (3%), Ty (10%), Asn (3%),
Th (3%), Me (7%), Phe (20%), T p (3%), Gln (7%) and His (3%). In his g oup 47%
a e cha ged, 23% pola and 30% non-pola . Two HS conside a ions we e made: (i)
when he minimum ee binding ene gy upon alanine mu a ion was conside ed 2.0
kcal/mol: we had 23% o HS and 77% o NS and (ii) when he minimum ee binding
ene gy alue upon alanine mu a ion was conside ed 1.0 kcal/mol we had 37% as HS
and 63% os NS. All esidue choices a e limi ed o he exis ence o expe imen al ee
binding ene gy alues upon alanine mu a ion in he p o ein-based in e aces s udied.
3.1 - RMSD
RMSD p o iles we e calcula ed o each o he sys ems, conside ing sepa a ely
he p o ein and he nucleic acid con ibu ion, o assu e hei equilib a ion h oughou he
MD simula ion. All six complexes we e s able h oughou he MD simula ion wi h
a ia ions lowe han 2 Å in he DNA-based complexes and 3 Å in he RNA-based
complexes.
FCUP
S udy o P o ein-nucleic acid Complexes
41
48
FCUP
S udy o P o ein-nucleic acid Complexes
1URN
M
0.5400
-42.47
±
4.29
-0.68
±
0.07
Q
4.8500
-30.24
±
4.88
-0.87
±
0.04
F
3.2300
-63.25
±
5.71
-0.92
±
0.03
2ERR
H
2.9800
-31.58
±
5.58
-0.70
±
0.12
F
4.3100
-111.90
±
6.38
-0.75
±
0.07
F
3.8700
-53.51
±
7.09
-0.85
±
0.08
F
6.0800
-51.06
±
7.74
-0.96
±
0.04
Table 10 – Resul s o ∆SASA and elSASA o all esidues wi h known ∆∆Gbinding o he s udied complexes.
Since he ype o amino acid plays a c ucial ole in he de ini ion o he in e ace,
hey we e g ouped acco ding o hei chemical cha ac e : cha ged (Glu, His, Lys and
A g); Pola (Th , Asn, Gln and Ty ) and nonpola (Me , Phe and T p).
Figu e 12 - G aphical Rep esen a ion o a e age ∆SASA alues o each o he amino acid g oups o he 2.0 kcal/mol
cu -o .
Figu e 13 - G aphical Rep esen a ion o a e age elSASA alues o each o he amino acid g oups o he 2.0 kcal/mol
cu -o .
0,00
20,00
40,00
60,00
80,00
All Cha ged Pola Non Pola
∆ SASA / A2
Ho -Spo s
Null-Spo s
0
0,2
0,4
0,6
0,8
1
All Cha ged Pola Non Pola
el SASA
Ho -Spo s
Null-Spo s

FCUP
S udy o P o ein-nucleic acid Complexes
49
Fo an easie analysis o he esul s p esen ed in Table 10, hey we e plo ed in
2 g aphics and shown in Figu es 11 and 12. The SASA analysis by i sel is insu icien
o make a clea dis inc ion be ween HS and NS as we can see in Figu e 11, (bo h HS
and NS ha e high alues o ∆SASA). The a e age alue o HS is 56.39 ± 6.40 Å2
(∆SASA) and 0.81 ± 0.07 ( elSASA) and 52.07 ± 7.15 Å2 (∆SASA) and 0.48 ± 0.06
( elSASA) o NS. Fo his 2.0 kcal/mol cu -o all h ee ca ego ies o NS ha e highe
alues o ∆SASA han he espec i e ones o HS despi e he o al a e age being highe
o he HS. The highe di e ence is in he cha ged g oup as NS ha e a ∆SASA alue o
52.10 ± 7.49 Å2, 10 Å2 mo e han he HS a e age, while in he pola and non-pola
g oup he di e ences a e 2 Å2 and 5 Å2 espec i ely. RelSASA alues don’ ollow his
endency and a e highe o all he conside ed HS g oups and each hei maximum
di e ence in he pola g oup whe e HS ha e 0.87 ± 0.04 and NS only 0.41 ± 0.07.
Figu e 14 - G aphical Rep esen a ion o a e age ∆SASA alues o each o he amino acid g oups o he 1.0 kcal/mol
cu -o .
Figu e 15 - G aphical Rep esen a ion o a e age elSASA alues o each o he amino acid g oups o he 1.0 kcal/mol
cu -o .
0,00
20,00
40,00
60,00
80,00
100,00
All Cha ged Pola Non Pola
∆ SASA / A2
Ho -Spo s
Null-Spo s
0
0,2
0,4
0,6
0,8
1
All Cha ged Pola Non Pola
el SASA
Ho -Spo s
Null-Spo s
50
FCUP
S udy o P o ein-nucleic acid Complexes
The esul s o he 1.0 kcal/mol cu -o a e plo ed in Figu es 13 and 14. In his
case he HS o he cha ged g oup has a much lowe alue o ∆SASA (60.02 ± 7.38 Å2)
while he NS a e age is only 45.53 ± 7.33 Å2 making a di e ence o oughly 15 Å2. The
pola g oup emains e y close wi h a di e ence o only 2 Å2 and he non-pola g oup is
he one wi h he bigge di e ence in ∆SASA alues, 91.54 ± 7.63 Å2 o NS and 58.49
± 6.15 Å2 o HS. Fo he elSASA alues is no o ious a big di e ence (double)
be ween HS and NS whe e HS ha e a highe alue bu when compa ing he non-pola
g oup he alue is he same, 0.81 ± 0.08 o HS and 0.81 ± 0.04 o NS.
To y o unde s and he possible di e ences be ween DNA and RNA-based
complexes, i was made a sepa a ely analysis o ∆SASA and elSASA as well as o
he 2.0 kcal/mol and 1.0 kcal/mol cu -o s.
Figu e 16 - G aphical Rep esen a ion o a e age ∆SASA alues o each o he amino acid g oups o he 2.0 kcal/mol
cu -o .
Figu e 17 - G aphical Rep esen a ion o a e age elSASA alues o each o he amino acid g oups o he 2.0 kcal/mol
cu -o .
0,00
20,00
40,00
60,00
80,00
100,00
All Cha ged Pola Non Pola
∆ SASA / A2
Ho -Spo s
Null-Spo s
0
0,2
0,4
0,6
0,8
1
All Cha ged Pola Non Pola
el SASA
Ho -Spo s
Null-Spo s
FCUP
S udy o P o ein-nucleic acid Complexes
51
The esul s o he DNA-based complexes 2.0 kcal/mol cu -o a e plo ed in
Figu es 15 and 16. This sepa a e analysis is poo e han he global one due o he lack
o HS in he pola and non-pola g oup emaining only he cha ged g oup. In his g oup
we can obse e ha HS ha e a sligh ly highe alue o ∆SASA han NS and a bigge
di e ence o elSASA 53.19 ± 7.39 Å2; 0.66 ± 0.09 (HS) and 52.10 ± 7.49 Å2; 0.39 ±
0.06 (NS).
Figu e 18 - G aphical Rep esen a ion o a e age ∆SASA alues o each o he amino acid g oups o he 1.0 kcal/moll
cu -o .
Figu e 19 - G aphical Rep esen a ion o a e age elSASA alues o each o he amino acid g oups o he 1.0 kcal/mol
cu -o .
The esul s o he 1.0 kcal/mol cu -o a e plo ed in Figu es 17 and 18. Once
again he e a e no HS in he pola g oup so he compa ison can only be made o he
cha ged and non-pola g oups. In he Cha ged g oup HS ha e a highe alue o
∆SASA and elSASA, 67.14 ± 7.84 Å2; 0.65 ± 0.08 (HS) and 45.53 ± 7.33 Å2; 0.30 ±
0.05 (NS) bu in he non-pola g oup is he o he way a ound and he di e ence is
massi e in a ou o NS, 12.72 ± 3.8 Å2; 0.57 ± 0.16 (HS) and 107.9 ± 8.74 Å2; 0.85 ±
0,00
20,00
40,00
60,00
80,00
100,00
120,00
All Cha ged Pola Non Pola
∆ SASA / A2
Ho -Spo s
Null-Spo s
0
0,2
0,4
0,6
0,8
1
All Cha ged Pola Non Pola
el SASA
Ho -Spo s
Null-Spo s
52
FCUP
S udy o P o ein-nucleic acid Complexes
0.03 (NS). This huge esul is no no mal, bu can be explained wi h he small numbe
o non-pola esidues a hese in e aces and he size o hese complexes, making
hem ulne able o he sol en ac ion.
Figu e 20 - G aphical Rep esen a ion o a e age ∆SASA alues o each o he amino acid g oups o he 2.0 kcal/mol
cu -o .
Figu e 21 - G aphical Rep esen a ion o a e age el SASA alues o each o he amino acid g oups o he 2.0 kcal/mol
cu -o .
0,00
20,00
40,00
60,00
80,00
All Cha ged Pola Non Pola
∆ SASA / A2
Ho -Spo s
Null-Spo s
0
0,2
0,4
0,6
0,8
1
All Cha ged Pola Non Pola
el SASA
Ho -Spo s
Null-Spo s
FCUP
S udy o P o ein-nucleic acid Complexes
53
The esul s o ∆SASA and elSASA in he RNA-based complexes o he 2.0
kcal/mol cu -o a e plo ed in Figu es 19 and 20. I in he DNA-based complexes we
lacked HS, in RNA-based complexes we lack NS, and so in his cu -o he only
possible compa ison is he non-pola g oup which has an opposi e esul when
compa ed wi h he p e ious analysis. In his non-pola g oup HS ha e a highe alue o
∆SASA and elSASA, 69.93 ± 6.73 Å2; 0.87 ± 0.06 (HS) and 42.47 ± 4.29 Å2; 0.68 ±
0.07 (NS).
Fo he 1.0 kcal/mol cu -o is an analysis is no equi ed because he e is no
change in he ∆SASA and elSASA alues.
3.4 - Mu agenesis in P o ein acid nucleic in e aces
We ha e applied he ASM me hodology i s in i s o iginal o mula ion, using he
LPB equa ion o calcula e he Gpola sol a ion e m. Fo ha we calcula ed he ∆∆Gbinding
alue o a se o 25 s uc u es gene a ed om he las 2 ns o MD simula ions in
explici sol en . Th oughou his wo k we pe o med he a ious calcula ions wi h
dielec ic cons an s anged om ɛ1 o ɛ9 o access he impo ance o he dielec ic
cons an in he de e mina ion. As he esul s we e no accu a e enough we will no
p esen hem in his hesis. Then we used he NLPB equa ion ins ead o he LPB
implemen ed in he DelPhi p og am o he calcula ion o he binding ee ene gy upon
alanine mu a ion. This was done because i was p o ed ha using he LPB equa ion is
no he mos app op ia e me hod o dealing wi h highly cha ged sys ems such as he
P o ein-nucleic acid sys ems in s udy. Ha ing his in mind only he esul s a e he use
o he NLPB we e ea ed and a e shown in Table 11( om ɛ1 o ɛ4) and Table 12( om
ɛ5 o ɛ9). We also ha e o s ess ou ha ins ead o p esen ing ∆∆Gbinding, we will
p esen ∆∆Gpola sol + ∆∆ɛelec ic as i show o co ela e mo e accu a ely wi h he
expe imen al alues.

54
FCUP
S udy o P o ein-nucleic acid Complexes
AA
Mu ed
∆∆Gexp
/
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
ɛ1
ɛ2
ɛ3
ɛ4
1ECR
R
1,2000
-8,08
0,31
3,02
4,32
Q
0,1100
-3,84
-1,89
-1,24
-0,91
1J5N
K
0,4300
3,33
5,11
5,69
5,98
R
0,6300
-8,25
1,51
4,58
6,03
Y
0,9000
-1,16
-0,56
-0,30
-0,16
M
0,4600
-1,77
-0,88
-0,57
-0,42
N
0,7400
-3,25
-1,32
-0,68
-0,37
R
0,8400
2,23
4,86
5,72
6,14
R
0,7200
-5,43
1,09
3,21
4,24
F
0,4000
-4,26
-2,10
-1,37
-1,01
K
0,4900
-6,96
0,08
2,42
3,58
K
0,0000
-0,11
2,21
3,00
3,40
K
0,0000
-0,32
1,93
2,70
3,09
K
0,4000
-3,01
1,62
3,15
3,92
K
0,2500
0,43
3,57
4,59
5,09
K
0,4300
-2,36
2,63
4,25
5,06
Y
0,2100
-0,86
-0,32
-0,16
-0,08
Y
0,3600
-0,99
-0,40
-0,21
-0,12
1JMC
R
2,1500
7,14
6,51
5,95
5,56
F
0,1900
-2,70
-1,31
-0,84
-0,61
E
1,3600
1,34
-0,26
-0,86
-1,19
W
1,0900
-0,50
-0,10
0,05
0,11
R
1,9100
6,84
6,63
6,46
6,29
1TN9
T
0,0800
-3,96
-1,95
-1,28
-0,94
1URN
M
0,5400
-2,78
-1,19
-0,70
-0,47
Q
4,8500
5,91
2,85
1,82
1,31
F
3,2300
-2,68
-1,16
-0,71
-0,49
2ERR
H
2,9800
-4,88
-2,30
-1,46
-1,05
F
4,3100
-5,40
-2,60
-1,68
-1,22
F
3,8700
-5,77
-2,69
-1,68
-1,19
F
6,0800
-2,11
-1,01
-0,65
-0,47
Table 11 - MM-PBSA esul s o ɛ1 o ɛ4
FCUP
S udy o P o ein-nucleic acid Complexes
55
AA
Mu ed
∆∆Gexp /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
ɛ5
ɛ6
ɛ7
ɛ8
ɛ9
1ECR
R
1,2000
5,08
5,56
5,90
6,14
6,31
Q
0,1100
-0,71
-0,58
-0,49
-0,42
-0,37
1J5N
K
0,4300
6,16
6,27
37,06
6,40
6,45
R
0,6300
6,85
7,35
7,69
7,93
8,10
Y
0,9000
-0,08
-0,03
0,01
0,03
0,05
M
0,4600
-0,33
-0,27
-0,23
-0,20
-0,17
N
0,7400
-0,19
-0,07
0,02
0,08
0,12
R
0,8400
6,38
6,54
6,65
6,73
6,79
R
0,7200
4,84
5,22
5,48
5,67
5,81
F
0,4000
-0,79
-0,64
-0,54
-0,46
-0,40
K
0,0000
3,65
3,82
3,95
4,05
4,13
K
0,0000
3,34
3,51
3,63
3,73
3,81
K
0,4000
4,37
4,68
4,89
5,05
5,17
K
0,2500
5,38
5,56
5,69
5,78
5,84
K
0,4300
5,53
5,85
6,06
6,22
6,35
Y
0,2100
-0,03
0,00
0,02
0,03
0,04
Y
0,3600
-0,07
-0,03
-0,01
0,01
0,02
1JMC
R
2,1500
5,27
5,03
4,82
4,64
4,49
F
0,1900
-0,48
-0,38
-0,32
-0,27
-0,23
E
1,3600
-1,39
-1,54
-1,64
-1,72
-1,78
W
1,0900
0,14
0,16
0,17
0,18
0,18
R
1,9100
6,13
5,99
5,86
5,74
5,64
1TN9
T
0,0800
-0,57
-0,47
-0,39
-0,34
-0,29
1URN
M
0,5400
-0,34
-0,26
-0,21
-0,17
-0,14
Q
4,8500
1,01
0,81
0,67
0,56
0,48
F
3,2300
-0,37
-0,29
-0,24
-0,20
-0,17
2ERR
H
2,9800
-0,81
-0,66
-0,55
-0,47
-0,41
F
4,3100
-0,95
-0,77
-0,65
-0,55
-0,48
F
3,8700
-0,86
-0,71
-0,58
-0,49
-0,41
F
6,0800
-0,36
-0,29
-0,24
-0,20
-0,18
Table 12 - MM-PBSA esul s o ɛ5 o ɛ9
The a e age e o s o he calcula ed alues o ∆∆Gpola sol + ∆∆ɛele ene gy a e
shown in Table 13, whe e hey we e sepa a ed in he h ee: cha ged, pola and non-
pola . This alone is no enough o in e abou he applicabili y o he me hod and i s
accu acy, so a s a is ical analysis is also necessa y and will be analysed by a se o
es s: (i) F1 sco e (equa ion 17) de ined as a unc ion o P ecision (P, equa ion 18),
which indica es he eliabili y o he p edic ions and he ou come o alanine mu a ions;
and (ii) Recall which is ela ed wi h he numbe o HS co ec ly p edic ed and he e o e
is c ucial in hese s udies (R, equa ion 19). TP s ands o ue posi i e (p edic ed HS
56
FCUP
S udy o P o ein-nucleic acid Complexes
ha a e ac ual HS) and FP s ands o alse posi i e (p edic ed HS ha a e no an ac ual
HS), TN s ands o ue nega i e (p edic ed NS ha a e ac ual NS) and FN s ands o
alse nega i e (p edic ed NS ha a e no ac ual NS). Speci ici y (equa ion 20) is
ano he measu e o pe o mance, especially o NS. F1 and Accu acy (equa ion 21)
gi e he o e all pe o mance o he me hods, so he ideal me hod would ha e hese
alues as close o 100% as possible.
Fo a be e display and discussion, his s a is ical analysis will be p esen ed
sepa a ely o he 2.0 kcal/moll cu -o and 1.0 kcal/mol cu -o .
|∆∆GMM-PBSA - ∆∆Gexp| kcal/mol
1
2
3
4
5
6
7
8
9
All
3,96
2,72
2,92
3,04
3,10
3,14
4,19
3,18
3,19
Cha ged
3,45
2,49
3,42
3,92
4,21
4,39
6,70
4,59
4,65
Pola
3,17
2,02
1,72
1,57
1,46
1,41
1,37
1,34
1,32
Non-pola
5,38
3,72
3,18
2,91
2,75
2,66
2,59
2,53
2,49
Table 13 - Resul s o a e age e o s ob ained wi h he NLPB equa ion.
In Table 13 a e displayed he a e age e o s ob ained o each dielec ic
cons an , globally and o each g oup o esidues. The a e age e o alue goes om
1.32 kcal/mol wi h ɛ9 o he pola g oup o 5.38 kcal/mol wi h ɛ1 o he non-pola
g oup ( he o al a e age o e o a e age is 3.08 kcal/mol). Ha ing hese e o alues in
conside a ion, he bes dielec ic cons an o e all is ɛ2 wi h an a e age e o o 2.72
kcal/mol; o he cha ged g oups, ɛ2 is also he bes wi h an a e age e o o 2.49
kcal/mol; o he pola and non-pola g oups he bes dielec ic cons an is also ɛ2 wi h
a e age e o s o 2.02 kcal/mol and 3.72 kcal/mol espec i ely. Al hough ɛ9 alues
FCUP
S udy o P o ein-nucleic acid Complexes
57
we e be e (1.32 kcal/mol and 2.49 kcal/mol espec i ely), hey we e missing a
physical explana ion and he e o e we e excluded.
S a is ical es s/all
ɛ1
ɛ 2
ɛ 3
ɛ 4
ɛ 5
ɛ 6
ɛ 7
ɛ 8
ɛ 9
P
33.3
41.7
8.3
8.3
8.3
8.3
8.3
8.3
8.3
R
100.0
71.4
14.3
14.3
14.3
14.3
14.3
14.3
14.3
F1
50.0
52.6
10.5
10.5
10.5
10.5
10.5
10.5
10.5
Accu acy
53.3
70.0
43.3
43.3
43.3
43.3
43.3
43.3
43.3
Speci ici y
39.1
69.6
52.2
52.2
52.2
52.2
52.2
52.2
52.2
S a is ical es s/cha ged
ɛ1
ɛ 2
ɛ 3
ɛ 4
ɛ 5
ɛ 6
ɛ 7
ɛ 8
ɛ 9
P
20.0
25.0
8.3
8.3
8.3
8.3
8.3
8.3
8.3
R
100.0
100.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
F1
33.3
40.0
14.3
14.3
14.3
14.3
14.3
14.3
14.3
Accu acy
42.9
57.1
14.3
14.3
14.3
14.3
14.3
14.3
14.3
Speci ici y
33.3
50.0
8.3
8.3
8.3
8.3
8.3
8.3
8.3
S a is ical es s/pola
ɛ1
ɛ 2
ɛ 3
ɛ 4
ɛ 5
ɛ 6
ɛ 7
ɛ 8
ɛ 9
P
25.0
100.0
-
-
-
-
-
-
-
R
100.0
100.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
F1
40.0
100.0
-
-
-
-
-
-
-
Accu acy
57.1
100.0
85.7
85.7
85.7
85.7
85.7
85.7
85.7
Speci ici y
50.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
S a is ical es s/non-pola
ɛ1
ɛ 2
ɛ 3
ɛ 4
ɛ 5
ɛ 6
ɛ 7
ɛ 8
ɛ 9
P
57.1
66.7
-
-
-
-
-
-
-
R
100.0
50.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
F1
72.7
57.1
-
-
-
-
-
-
-
Accu acy
66.7
66.7
55.6
55.6
55.6
55.6
55.6
55.6
55.6
Speci ici y
40.0
80.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
Table 14 - Resul s o S a is ical es s o he 2.0 kcal/mol cu -o
In Table 14 a e p esen ed he s a is ical esul s o he 2.0 kcal/mol cu -o . Fo
an easie eading, he discussion will be made in e ms o HS p edic ion accu acy
o e all and o each o he g oups conside ed (cha ged, pola and non-pola ), o each
dielec ic cons an . O e all, he e a e 7HS o a o al o 30 esidues, whe e 1HS
belongs o DNA-based complexes and he o he 6 o RNA-based complexes. The
cha ged g oup has 14 esidues, he pola g oup 7 and he non-pola has 9.
64
FCUP
S udy o P o ein-nucleic acid Complexes
p edic ed o all dielec ic cons an s was a yp ophan (T p) which is also he only one
o he da a se . ɛ1 showed he bigge numbe o FP again and he ɛ2 he lowe bu ɛ2
ailed o p edic 4/11 HS, whe e 3 o hem belong o DNA-based complexes. In his cu -
o HS om RNA-based complexes we e co ec ly p edic ed wi h an accu acy o 100%
o ɛ1, ɛ2 (83%), ɛ3 (67%), ɛ4 (67%), ɛ5 (16%) and alling o 0% om ɛ6 o ɛ9.
Conside ing he ɛ2, i was once again he one wi h he leas FP, only 6 (again hal om
ɛ1). HS p edic ion was be e in RNA-based complexes wi h 5/6 bu wo se in DNA-
based complexes wi h 2/4 co ec ly p edic ed HS. Conside ing he ɛ9, which is
s a is ically iden ical o ɛ6, ɛ7 and ɛ8 wi h he same exac numbe o TP, FN, TP and
FN, all ailing o iden i y a single HS in he RNA-based complexes despi e he 80%
(4/5) HS p edic ion in DNA-based complexes.
The majo p oblem o bo h cu -o s was he cha ged g oup o amino acids
which has 100% o he FP o all da a se when conside ing ɛ3 o ɛ9. In he o he
g oups he e is only 1 FP in ɛ2 in he non-pola g oup and 6 in ɛ1 dis ibu ed 3 each in
he pola and non-pola g oup.
Figu e 22 - Schema ic ep esen a ion o he inal me hod o mula ion o he de e mina ion o ∆∆Gbinding
Wild- ype s uc u e
MD simula ion wi h explici sol en
ep esen a ion
ASM calcula ion and
analysis using
elec os a ic and pola
sol a ion e ms Applica ion o he NLPB
equa ion
ɛ2 o all amino acids
∆∆Gbinding

FCUP
S udy o P o ein-nucleic acid Complexes
65
Re e ences
1. Janin, J., ELUSIVE AFFINITIES. P o eins-S uc u e Func ion and Gene ics,
1995. 21(1): p. 30-39.
2. Jones, S. and J.M. Tho n on, P inciples o p o ein-p o ein in e ac ions.
P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o
Ame ica, 1996. 93(1): p. 13-20.
3. Cho hia, C. and J. Janin, PRINCIPLES OF PROTEIN-PROTEIN
RECOGNITION. Na u e, 1975. 256(5520): p. 705-708.
4. Clackson, T., e al., S uc u al and unc ional analysis o he 1 : 1 g ow h
ho mone : ecep o complex e eals he molecula basis o ecep o a ini y.
Jou nal o Molecula Biology, 1998. 277(5): p. 1111-1128.
5. DeLano, W.L., e al., Con e gen solu ions o binding a a p o ein-p o ein
in e ace. Science, 2000. 287(5456): p. 1279-1283.
6. DeLano, W.L., Un a eling ho spo s in binding in e aces: p og ess and
challenges. Cu en Opinion in S uc u al Biology, 2002. 12(1): p. 14-20.
7. Tho n, K.S. and A.A. Bogan, ASEdb: a da abase o alanine mu a ions and hei
e ec s on he ee ene gy o binding in p o ein in e ac ions. Bioin o ma ics,
2001. 17(3): p. 284-285.
8. Mo ei a, I.S., P.A. Fe nandes, and M.J. Ramos, Ho spo s-A e iew o he
p o ein-p o ein in e ace de e minan amino-acid esidues. P o eins-S uc u e
Func ion and Bioin o ma ics, 2007. 68(4): p. 803-812.
9. Mo ei a, I.S., P.A. Fe nandes, and M.J. Ramos, Un a eling he impo ance o
p o ein-p o ein in e ac ion: Applica ion o a compu a ional alanine-scanning
mu agenesis o he s udy o he IgG1 s ep ococcal p o ein G (C2 agmen )
complex. Jou nal o Physical Chemis y B, 2006. 110(22): p. 10962-10969.
10. Mo ei a, I.S., P.A. Fe nandes, and M.J. Ramos, De ailed mic oscopic s udy o
he ull ZipA : F sZ in e ace. P o eins-S uc u e Func ion and Bioin o ma ics,
2006. 63(4): p. 811-821.
11. Mo ei a, I.S., P.A. Fe nandes, and M.J. Ramos, Compu a ional alanine
scanning mu agenesis - An imp o ed me hodological app oach. Jou nal o
Compu a ional Chemis y, 2007. 28(3): p. 644-654.
12. Huo, S., I. Masso a, and P.A. Kollman, Compu a ional alanine scanning o he
1 : 1 human g ow h ho mone- ecep o complex. Jou nal o Compu a ional
Chemis y, 2002. 23(1): p. 15-27.
13. Masso a, I. and P.A. Kollman, Compu a ional alanine scanning o p obe
p o ein-p o ein in e ac ions: A no el app oach o e alua e binding ee ene gies.
Jou nal o he Ame ican Chemical Socie y, 1999. 121(36): p. 8133-8143.
14. Chak aba i, P. and J. Janin, Dissec ing p o ein-p o ein ecogni ion si es.
P o eins-S uc u e Func ion and Gene ics, 2002. 47(3): p. 334-343.
15. Bahadu , R.P., e al., Dissec ing subuni in e aces in homodime ic p o eins.
P o eins-S uc u e Func ion and Gene ics, 2003. 53(3): p. 708-719.
16. Guha oy, M. and P. Chak aba i, Conse a ion and ela i e impo ance o
esidues ac oss p o ein-p o ein in e aces. P oceedings o he Na ional
Academy o Sciences o he Uni ed S a es o Ame ica, 2005. 102(43): p. 15447-
15452.
17. Bogan, A.A. and K.S. Tho n, Ana omy o ho spo s in p o ein in e aces. J Mol
Biol, 1998. 280(1): p. 1-9.
18. Li, J. and Q. Liu, 'Double wa e exclusion': a hypo hesis e ining he O- ing
heo y o he ho spo s a p o ein in e aces. Bioin o ma ics, 2009. 25(6): p.
743-750.
66
FCUP
S udy o P o ein-nucleic acid Complexes
19. Koslo , M., e al., In eg a ing ene gy calcula ions wi h unc ional assays o
deciphe he speci ici y o G p o ein-RGS p o ein in e ac ions. Na u e S uc u al
& Molecula Biology, 2011. 18(7): p. 846-U128.
20. Rajamani, D., e al., Ancho esidues in p o ein-p o ein in e ac ions.
P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o
Ame ica, 2004. 101(31): p. 11287-11292.
21. Mo ei a, I.S., P.A. Fe nandes, and M.J. Ramos, Ho spo occlusion om bulk
wa e : A comp ehensi e s udy o he complex be ween he lysozyme HEL and
he an ibody FVD1.3. Jou nal o Physical Chemis y B, 2007. 111(10): p. 2697-
2706.
22. Ahmad, S., e al., P o ein-DNA in e ac ions: s uc u al, he modynamic and
clus e ing pa e ns o conse ed esidues in DNA-binding p o eins. Nucleic
Acids Resea ch, 2008. 36(18): p. 5922-5932.
23. B adshaw, R.T., e al., Compa ing expe imen al and compu a ional alanine
scanning echniques o p obing a p o o ypical p o ein-p o ein in e ac ion.
P o ein Eng Des Sel, 2011. 24(1-2): p. 197-207.
24. Ze be, B.S., e al., Rela ionship be ween ho spo esidues and ligand binding
ho spo s in p o ein-p o ein in e aces. J Chem In Model, 2012. 52(8): p. 2236-
44.
25. Guha oy, M., e al., PRICE (PRo ein In e ace Conse a ion and Ene ge ics): a
se e o he analysis o p o ein-p o ein in e aces. J S uc Func Genomics,
2011. 12(1): p. 33-41.
26. Mo ei a, I.S., P.A. Fe nandes, and M.J. Ramos, Un a eling he impo ance o
p o ein-p o ein in e ac ion: applica ion o a compu a ional alanine-scanning
mu agenesis o he s udy o he IgG1 s ep ococcal p o ein G (C2 agmen )
complex. J Phys Chem B, 2006. 110(22): p. 10962-9.
27. Law, M.J., e al., The ole o posi i ely cha ged amino acids and elec os a ic
in e ac ions in he complex o U1A p o ein and U1 hai pin II RNA. Nucleic Acids
Res, 2006. 34(1): p. 275-85.
28. Be man, H.M., e al., The P o ein Da a Bank. Nucleic Acids Res, 2000. 28(1): p.
235-42.
29. Kuma , M.D., e al., P oThe m and P oNIT: he modynamic da abases o
p o eins and p o ein-nucleic acid in e ac ions. Nucleic Acids Res, 2006.
34(Da abase issue): p. D204-6.
30. P abaka an, P., e al., The modynamic da abase o p o ein-nucleic acid
in e ac ions (P oNIT). Bioin o ma ics, 2001. 17(11): p. 1027-34.
31. Sa ai, A., [The modynamic da abases o p o eins and in e ac ions].
Tanpakushi su Kakusan Koso, 2002. 47(8 Suppl): p. 1071-5.
32. Ho man, M.M., e al., AANT: he Amino Acid-Nucleo ide In e ac ion Da abase.
Nucleic Acids Res, 2004. 32(Da abase issue): p. D174-81.
33. Luscombe, N.M., e al., An o e iew o he s uc u es o p o ein-DNA
complexes. Genome Biol, 2000. 1(1): p. REVIEWS001.
34. No ambuena, T. and F. Melo, The P o ein-DNA In e ace da abase. BMC
Bioin o ma ics, 2010. 11: p. 262.
35. Kamada, K., e al., S uc u e o a eplica ion- e mina o p o ein complexed wi h
DNA. Na u e, 1996. 383(6601): p. 598-603.
36. Kaplan, D.L. and D. Bas ia, Mechanisms o pola a es o a eplica ion o k. Mol
Mic obiol, 2009. 72(2): p. 279-85.
37. Bas ia, D., e al., Replica ion e mina ion mechanism as e ealed by Tus-
media ed pola a es o a sliding helicase. P oc Na l Acad Sci U S A, 2008.
105(35): p. 12831-6.
38. Neylon, C., e al., In e ac ion o he Esche ichia coli eplica ion e mina o
p o ein (Tus) wi h DNA: a model de i ed om DNA-binding s udies o mu an
FCUP
S udy o P o ein-nucleic acid Complexes
67
p o eins by su ace plasmon esonance. Biochemis y, 2000. 39(39): p. 11989-
99.
39. Masse, J.E., e al., The S. ce e isiae a chi ec u al HMGB p o ein NHP6A
complexed wi h DNA: DNA and p o ein con o ma ional changes upon binding. J
Mol Biol, 2002. 323(2): p. 263-84.
40. Allain, F.H., e al., Solu ion s uc u e o he HMG p o ein NHP6A and i s
in e ac ion wi h DNA e eals he s uc u al de e minan s o non-sequence-
speci ic binding. EMBO J, 1999. 18(9): p. 2563-79.
41. Coa s, J.E., e al., Single-molecule FRET analysis o DNA binding and bending
by yeas HMGB p o ein Nhp6A. Nucleic Acids Res, 2013. 41(2): p. 1372-81.
42. Zhang, J., e al., Basic N- e minus o yeas Nhp6A egula es he mechanism o
i s DNA lexibili y enhancemen . J Mol Biol, 2012. 416(1): p. 10-20.
43. McCauley, M., e al., Dual binding modes o an HMG domain om human
HMGB2 on DNA. Biophys J, 2005. 89(1): p. 353-64.
44. Bochka e , A., e al., S uc u e o he single-s anded-DNA-binding domain o
eplica ion p o ein A bound o DNA. Na u e, 1997. 385(6612): p. 176-81.
45. Wal he , A.P., e al., Replica ion p o ein A in e ac ions wi h DNA. 1. Func ions o
he DNA-binding and zinc- inge domains o he 70-kDa subuni . Biochemis y,
1999. 38(13): p. 3963-73.
46. B osey, C.A., e al., A new s uc u al amewo k o in eg a ing eplica ion
p o ein A in o DNA p ocessing machine y. Nucleic Acids Res, 2013.
47. Wyka, I.M., e al., Replica ion p o ein A in e ac ions wi h DNA: di e en ial
binding o he co e domains and analysis o he DNA in e ac ion su ace.
Biochemis y, 2003. 42(44): p. 12909-18.
48. Ozawa, K., e al., Mapping o he 70 kDa, 34 kDa, and 11 kDa subuni genes o
he human mul ime ic single-s anded DNA binding p o ein (hSSB/RPA) o
ch omosome bands 17p13, 1p35-p36.1, and 7p21-p22. Cell S uc Func , 1993.
18(4): p. 221-30.
49. Wojciak, J.M., K.M. Connolly, and R.T. Clubb, NMR s uc u e o he Tn916
in eg ase-DNA complex. Na S uc Biol, 1999. 6(4): p. 366-73.
50. Yao, X.X., e al., Molecula dynamics s udy o DNA binding by INT-DBD unde
pola ized o ce ield. J Compu Chem, 2013.
51. Rajee , L., K. Malanowska, and J.F. Ga dne , Challenging a pa adigm: he ole
o DNA homology in y osine ecombinase eac ions. Mic obiol Mol Biol Re ,
2009. 73(2): p. 300-9.
52. Connolly, K.M., M. Iwaha a, and R.T. Clubb, Xis p o ein binding o he le a m
s imula es excision o conjuga i e ansposon Tn916. J Bac e iol, 2002. 184(8):
p. 2088-99.
53. Connolly, K.M., e al., Majo g oo e ecogni ion by h ee-s anded be a-shee s:
a ini y de e minan s and conse ed s uc u al ea u es. J Mol Biol, 2000.
300(4): p. 841-56.
54. Go e, A.A., A. Ca lisch, and I. Jelesa o , The ole o lexibili y and hyd a ion on
he sequence-speci ic DNA ecogni ion by he Tn916 in eg ase p o ein: a
molecula dynamics analysis. J Mol Recogni , 2004. 17(2): p. 120-31.
55. Ka samba, P.S., e al., Complex ole o he be a 2-be a 3 loop in he in e ac ion
o U1A wi h U1 hai pin II RNA. J Biol Chem, 2002. 277(36): p. 33267-74.
56. Ka samba, P.S., D.G. Myszka, and I.A. Lai d-O inga, Two unc ionally dis inc
s eps media e high a ini y binding o U1A p o ein o U1 hai pin II RNA. J Biol
Chem, 2001. 276(24): p. 21476-81.
57. Showal e , S.A. and K.B. Hall, Al e ing he RNA-binding mode o he U1A RBD1
p o ein. J Mol Biol, 2004. 335(2): p. 465-80.
58. Law, M.J., e al., Kine ic analysis o he ole o he y osine 13, phenylalanine 56
and glu amine 54 ne wo k in he U1A/U1 hai pin II in e ac ion. Nucleic Acids
Res, 2005. 33(9): p. 2917-28.
68
FCUP
S udy o P o ein-nucleic acid Complexes
59. Oub idge, C., e al., C ys al s uc u e a 1.92 A esolu ion o he RNA-binding
domain o he U1A spliceosomal p o ein complexed wi h an RNA hai pin.
Na u e, 1994. 372(6505): p. 432-8.
60. Auwe e , S.D., e al., Molecula basis o RNA ecogni ion by he human
al e na i e splicing ac o Fox-1. EMBO J, 2006. 25(1): p. 163-73.
61. Ku oyanagi, H., Fox-1 amily o RNA-binding p o eins. Cell Mol Li e Sci, 2009.
66(24): p. 3895-907.
62. Fogel, B.L., e al., RBFOX1 egula es bo h splicing and ansc ip ional ne wo ks
in human neu onal de elopmen . Hum Mol Gene , 2012. 21(19): p. 4171-86.
63. Bi el, C.L., e al., E idence ha "b ain-speci ic" FOX-1, FOX-2, and
nPTB al e na i ely spliced iso o ms a e p oduced in he lens. Cu Eye Res,
2011. 36(4): p. 321-7.
64. Damiano , A. and D.L. Black, Au o egula ion o Fox p o ein exp ession o
p oduce dominan nega i e splicing ac o s. RNA, 2010. 16(2): p. 405-16.
65. Allinge , N.L., M.T. T ibble, and Y. Yuh, And os e one. The s uc u e by o ce-
ield calcula ions. S e oids, 1975. 26(4): p. 398-406.
66. S ewa , E.L., e al., Molecula Mechanics (MM3) Calcula ions on Oxygen-
Con aining Phospho us (Coo dina ion IV) Compounds. J O g Chem, 1999.
64(15): p. 5350-5360.
67. Chen, K.H., e al., Molecula mechanics (MM4) s udy o amines. J Compu
Chem, 2007. 28(15): p. 2391-412.
68. B ooks, B.R., e al., CHARMM: he biomolecula simula ion p og am. J Compu
Chem, 2009. 30(10): p. 1545-614.
69. Chea ham, T.E., 3 d, P. Cieplak, and P.A. Kollman, A modi ied e sion o he
Co nell e al. o ce ield wi h imp o ed suga pucke phases and helical epea .
J Biomol S uc Dyn, 1999. 16(4): p. 845-62.
70. Schyman, P. and W.L. Jo gensen, Explo ing Adso p ion o Wa e and Ions on
Ca bon Su aces using a Pola izable Fo ce Field. J Phys Chem Le , 2013. 4(3):
p. 468-474.
71. Pappala do, M., e al., F ee ene gy pe u ba ion and molecula dynamics
calcula ions o coppe binding o azu in. J Compu Chem, 2003. 24(6): p. 779-
85.
72. G uden-Pa lo ic, M., S. G ubisic, and S.R. Nike ic, Con o ma ional analysis o
oc a- and e ab omo e aphenylpo phy ins and hei Ni(II) and Tb(III)
complexes. J Ino g Biochem, 2004. 98(8): p. 1293-302.
73. Kleinjung, J., e al., Implici Sol a ion Pa ame e s De i ed om Explici Wa e
Fo ces in La ge-Scale Molecula Dynamics Simula ions. J Chem Theo y
Compu , 2012. 8(7): p. 2391-2403.
74. Wang, J., e al., De elopmen and es ing o a gene al ambe o ce ield. J
Compu Chem, 2004. 25(9): p. 1157-74.
75. Case, D.A.P., D. A.; Caldwell, J. W.; Chea ham, T. E.; and J.R. Wang, W. S.;
Simme ling, C.; Da den, T.; Me z, K. M.; S an on, R. V.; Cheng, A.; Vincen , J.
J.; C owley, M.; Tsui, V.; Gohlke H.; Radme , R.; Duan, Y.; Pi e a J.; Masso a,
I.; Seibel, G. L.; Singh, U. C.; Weine , P.; Kollman, P. , Uni e si y o Cali o nia:
San F ancisco, 2002.
76. Coleman, T.G., H.C. Mesick, and R.L. Da by, Nume ical in eg a ion: a me hod
o imp o ing solu ion s abili y in models o he ci cula ion. Ann Biomed Eng,
1977. 5(4): p. 322-8.
77. William L. Jo gensen, J.C., and Je y D. Madu a Compa ison o simple
po en ial unc ions o simula ing liquid wa e . AIP - The jou nal o chemical
physics, 1983. 79.
78. Zou, H., e al., Molecula insigh in o he in e ac ion be ween IFABP and PA by
using MM-PBSA and alanine scanning me hods. J Phys Chem B, 2007.
111(30): p. 9104-13.
FCUP
S udy o P o ein-nucleic acid Complexes
69
79. Huo, S., I. Masso a, and P.A. Kollman, Compu a ional alanine scanning o he
1:1 human g ow h ho mone- ecep o complex. J Compu Chem, 2002. 23(1): p.
15-27.
80. Yang, L., e al., New-gene a ion ambe uni ed-a om o ce ield. J Phys Chem B,
2006. 110(26): p. 13166-76.
81. Wang, W. and P.A. Kollman, F ee ene gy calcula ions on dime s abili y o he
HIV p o ease using molecula dynamics and a con inuum sol en model. J Mol
Biol, 2000. 303(4): p. 567-82.
82. Bash o d, D. and D.A. Case, Gene alized bo n models o mac omolecula
sol a ion e ec s. Annu Re Phys Chem, 2000. 51: p. 129-52.
83. Onu ie , A., D. Bash o d, and D.A. Case, Explo ing p o ein na i e s a es and
la ge-scale con o ma ional changes wi h a modi ied gene alized bo n model.
P o eins, 2004. 55(2): p. 383-94.
84. Luo, R., L. Da id, and M.K. Gilson, Accele a ed Poisson-Bol zmann calcula ions
o s a ic and dynamic sys ems. J Compu Chem, 2002. 23(13): p. 1244-53.
85. Li, L., e al., DelPhi: a comp ehensi e sui e o DelPhi so wa e and associa ed
esou ces. BMC Biophys, 2012. 5: p. 9.
86. Deche chi, S., e al., Be ween algo i hm and model: di e en Molecula Su ace
de ini ions o he Poisson-Bol zmann based elec os a ic cha ac e iza ion o
biomolecules in solu ion. Commun Compu Phys, 2013. 13: p. 61-89.
87. Mo ei a, I.S., P.A. Fe nandes, and M.J. Ramos, Compu a ional alanine
scanning mu agenesis--an imp o ed me hodological app oach. J Compu
Chem, 2007. 28(3): p. 644-54.
88. Hui Li, A.D.R., and Jan H. Jensen, Ve y Fas Empi ical P edic ion and
In e p e a ion o P o ein pKa Values. P o eins, 2005: p. 61, 704-721. .
89. Delphine C. Bas, D.M.R., and Jan H. Jensen, Ve y Fas P edic ion and
Ra ionaliza ion o pKa Values o P o ein-Ligand Complexes. P o eins, 2008: p.
73, 765-783. .
90. Ros kowski, M., e al., G aphical analysis o pH-dependen p ope ies o
p o eins p edic ed using PROPKA. BMC S uc Biol, 2011. 11: p. 6.
91. Dolinsky, T.J., e al., PDB2PQR: an au oma ed pipeline o he se up o
Poisson-Bol zmann elec os a ics calcula ions. Nucleic Acids Res, 2004.
32(Web Se e issue): p. W665-7.
92. Zhang, X., e al., Applica ion o new mul i- esolu ion me hods o he
compa ison o biomolecula elec os a ic p ope ies in he absence o global
s uc u al simila i y. Mul iscale Model Simul, 2006. 5(4): p. 1196-1213.
93. P ice, D.J. and C.L. B ooks, 3 d, A modi ied TIP3P wa e po en ial o
simula ion wi h Ewald summa ion. J Chem Phys, 2004. 121(20): p. 10096-103.
94. Huggins, D.J., Co ela ions in liquid wa e o he TIP3P-Ewald, TIP4P-2005,
TIP5P-Ewald, and SWM4-NDP models. J Chem Phys, 2012. 136(6): p. 064518.
95. Ribei o JV, C.N., Mo ei a IS, Fe nandes PA, Ramos MJ, Compasm: An ambe -
md alanine scanning mu agenesis plug-in. Theo e ical Chemis y Accoun s - In
p ess, 2012.
96. Ramos, R.M., L.F. Fe nandes, and I.S. Mo ei a, Ex ending he applicabili y o
he O- ing heo y o p o ein-DNA complexes. Compu Biol Chem, 2013. 44: p.
31-9.

70
FCUP
S udy o P o ein-nucleic acid Complexes
Annexes
RDF
1ECR
ARG_194
GLN_246
[Å]
N H2O
[Å]
N H2O
0,0500
0,0000
0,0500
0,0000
1,0500
0,0000
1,0500
0,0000
2,0500
0,0000
2,0500
0,0000
3,0500
0,2960
3,0500
0,5360
4,0500
0,9720
4,0500
1,7780
5,0500
1,8290
5,0500
3,7540
6,0500
3,3210
6,0500
7,0820
7,0500
7,0500
7,0500
12,1140
8,0500
14,2150
8,0500
19,7530
9,0500
25,5180
9,0500
30,0570
1J5N
ARG_23
ARG_36
ARG_40
ASN_33
LYS_22
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
3,0500
0,6060
3,0500
1,5470
3,0500
1,2050
3,0500
0,6520
3,0500
2,5600
4,0500
2,0660
4,0500
6,9890
4,0500
5,1090
4,0500
4,0310
4,0500
6,0830
5,0500
4,2630
5,0500
13,7540
5,0500
9,4510
5,0500
8,9340
5,0500
12,6620
6,0500
7,3160
6,0500
23,1130
6,0500
16,1530
6,0500
15,8120
6,0500
21,8940
7,0500
11,8290
7,0500
36,6490
7,0500
25,7220
7,0500
26,5360
7,0500
33,3100
8,0500
20,2540
8,0500
54,4670
8,0500
37,6770
8,0500
41,0400
8,0500
47,7640
9,0500
33,3780
9,0500
76,6920
9,0500
53,8290
9,0500
59,1880
9,0500
65,6580
1J5N
LYS_53
LYS_54
LYS_58
LYS_60
LYS_67
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
3,0500
2,4980
3,0500
2,2120
3,0500
2,4540
3,0500
2,5700
3,0500
2,7240
4,0500
5,3120
4,0500
5,9650
4,0500
5,3810
4,0500
5,8120
4,0500
4,7260
5,0500
10,4440
5,0500
11,5920
5,0500
10,2150
5,0500
12,2920
5,0500
8,3210
6,0500
18,3690
6,0500
19,7200
6,0500
17,1380
6,0500
21,8010
6,0500
14,0660
7,0500
29,1590
7,0500
30,3980
7,0500
26,1360
7,0500
34,4010
7,0500
21,2350
8,0500
44,3530
8,0500
44,4740
8,0500
38,0100
8,0500
50,8240
8,0500
31,0680
9,0500
64,7320
9,0500
63,0460
9,0500
53,4370
9,0500
71,3840
9,0500
45,2710
FCUP
S udy o P o ein-nucleic acid Complexes
71
1J5N
LYS_78
MET_29
PHE_48
TYR_28
TYR_81
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
0,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
1,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
2,0500
0,0000
3,0500
2,1420
3,0500
0,0000
3,0500
0,0510
3,0500
0,0000
3,0500
0,0030
4,0500
5,6710
4,0500
0,0380
4,0500
2,6540
4,0500
0,0000
4,0500
0,8410
5,0500
11,8270
5,0500
0,4930
5,0500
6,6570
5,0500
0,0000
5,0500
3,2320
6,0500
19,9150
6,0500
1,7490
6,0500
12,7380
6,0500
0,0580
6,0500
6,8350
7,0500
30,2660
7,0500
4,0860
7,0500
21,7800
7,0500
0,8770
7,0500
12,4130
8,0500
42,6490
8,0500
8,8910
8,0500
33,4690
8,0500
3,9140
8,0500
21,9030
9,0500
57,3470
9,0500
17,0410
9,0500
49,5850
9,0500
10,4670
9,0500
33,9100
1J5N
1TN9
TYR_88
THR_13
[Å]
N H2O
[Å]
N H2O
0,0500
0,0000
0,0500
0,0000
1,0500
0,0000
1,0500
0,0000
2,0500
0,0000
2,0500
0,0000
3,0500
0,0140
3,0500
1.4590
4,0500
1,2310
4,0500
4.3270
5,0500
3,9040
5,0500
8.9290
6,0500
7,9640
6,0500
16.2560
7,0500
14,7230
7,0500
25.7380
8,0500
23,6650
8,0500
37.7960
9,0500
37,2930
9,0500
52.8430
1JMC
PHE_56
ARG_200
GLU_95
ARG_52
TRP_179
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
0,05
0
0,05
0
0,05
0
0,05
0
0,05
0
1,05
0
1,05
0
1,05
0
1,05
0
1,05
0
2,05
0
2,05
0
2,05
0
2,05
0
2,05
0
3,05
0,025
3,05
0,804
3,05
0,696
3,05
0,677
3,05
0
4,05
1,406
4,05
2,124
4,05
1,556
4,05
2,9
4,05
0,001
5,05
3,472
5,05
3,743
5,05
3,212
5,05
4,631
5,05
0,123
6,05
6,262
6,05
6,656
6,05
3,916
6,05
7,136
6,05
0,956
7,05
9,142
7,05
11,712
7,05
6,476
7,05
8,648
7,05
2,375
8,05
13,34
8,05
18,762
8,05
10,26
8,05
12,542
8,05
5,291
9,05
19,526
9,05
28,982
9,05
14,921
9,05
16,908
9,05
11,45
72
FCUP
S udy o P o ein-nucleic acid Complexes
1URn
MET_50
GLN_53
PHE_55
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
0,05
0
0,05
0
0,05
0
1,05
0
1,05
0
1,05
0
2,05
0
2,05
0
2,05
0
3,05
0,002
3,05
0
3,05
0
4,05
0,593
4,05
0,012
4,05
0
5,05
2,04
5,05
0,385
5,05
0,095
6,05
3,597
6,05
1,611
6,05
0,817
7,05
5,615
7,05
3,887
7,05
1,589
8,05
9,731
8,05
7,387
8,05
3,338
9,05
17,104
9,05
12,626
9,05
5,984
2ERR
PHE_52
PHE_18
PHE_50
HID_12
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
[Å]
N H2O
0,05
0
0,05
0
0,05
0
0,05
0
1,05
0
1,05
0
1,05
0
1,05
0
2,05
0
2,05
0
2,05
0
2,05
0
3,05
0,003
3,05
0,082
3,05
0,088
3,05
0,858
4,05
0,224
4,05
2,373
4,05
0,969
4,05
1,94
5,05
1,035
5,05
6,917
5,05
1,392
5,05
4,929
6,05
2,908
6,05
12,295
6,05
2,882
6,05
7,225
7,05
5,859
7,05
21,135
7,05
5,444
7,05
11,682
8,05
12,226
8,05
32,875
8,05
9,697
8,05
18,701
9,05
22,528
9,05
47,613
9,05
18,405
9,05
27,391
FCUP
S udy o P o ein-nucleic acid Complexes
73
MM-PBSA
ɛ1
ɛ2
ɛ3
AA Mu ed
∆∆Gexp /
[kcal/mol]
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gabs
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gabs
∆∆Gpola
sol +
∆∆ɛele /
[kcal/mol]
∆∆Gabs
1ECR
R
1,2000
-8,08
9,28
0,31
0,89
3,02
1,82
Q
0,1100
-3,84
3,95
-1,89
2,00
-1,24
1,35
1J5N
K
0,4300
3,33
2,90
5,11
4,68
5,69
5,26
R
0,6300
-8,25
8,88
1,51
0,88
4,58
3,95
Y
0,9000
-1,16
2,06
-0,56
1,46
-0,30
1,20
M
0,4600
-1,77
2,23
-0,88
1,34
-0,57
1,03
N
0,7400
-3,25
3,99
-1,32
2,06
-0,68
1,42
R
0,8400
2,23
1,39
4,86
4,02
5,72
4,88
R
0,7200
-5,43
6,15
1,09
0,37
3,21
2,49
F
0,4000
-4,26
4,66
-2,10
2,50
-1,37
1,77
K
0,0000
-0,11
0,11
2,21
2,21
3,00
3,00
K
0,0000
-0,32
0,32
1,93
1,93
2,70
2,70
K
0,4000
-3,01
3,41
1,62
1,22
3,15
2,75
K
0,2500
0,43
0,18
3,57
3,32
4,59
4,34
K
0,4300
-2,36
2,79
2,63
2,20
4,25
3,82
Y
0,2100
-0,86
1,07
-0,32
0,53
-0,16
0,37
Y
0,3600
-0,99
1,35
-0,40
0,76
-0,21
0,57
1JMC
R
2,1500
7,14
4,99
6,51
4,36
5,95
3,80
F
0,1900
-2,70
2,89
-1,31
1,50
-0,84
1,03
E
1,3600
1,34
0,02
-0,26
1,62
-0,86
2,22
W
1,0900
-0,50
1,59
-0,10
1,19
0,05
1,04
R
1,9100
6,84
4,93
6,63
4,72
6,46
4,55
1TN9
T
0,0800
-3,96
4,04
-1,95
2,03
-1,28
1,36
1URN
M
0,5400
-2,78
3,32
-1,19
1,73
-0,70
1,24
Q
4,8500
5,91
1,06
2,85
2,00
1,82
3,03
F
3,2300
-2,68
5,91
-1,16
4,39
-0,71
3,94
2ERR
H
2,9800
-4,88
7,86
-2,30
5,28
-1,46
4,44
F
4,3100
-5,40
9,71
-2,60
6,91
-1,68
5,99
F
3,8700
-5,77
9,64
-2,69
6,56
-1,68
5,55
F
6,0800
-2,11
8,19
-1,01
7,09
-0,65
6,73
34
R.M.
Ramos
e
al.
/
Compu a ional
Biology
and
Chemis y
44
(2013)
31–39
Table
2
Desc ip ion
o
he
112
esidues
ha
cons i u e
ou
da ase ,
e idencing
he
espec i e
sys em,
PDB
nume a ion,
amino
acid
ype
and
expe imen ally
Gbinding.
P o ein
ID
#AA
PDB
#AA
mu a ed
Gbinding/
kcal
mol−1
Re e ence
1MNM
16
K
0.54 Ac on
e
al.
(2000)
1MNM
17
E
0.24
1MNM 18 R 0.28
1MNM
20
K
0.64
1MNM 21
I
0.88
1MNM
22
E
0.20
1MNM
23
I
0.86
1MNM
24
K
−0.13
1MNM 25 F 4.05
1MNM 26 I 4.05
1MNM
27
E
0.41
1MNM
28
N
1.75
1MNM
29
K
−0.13
1MNM
32
R
2.76
1MNM
33
H
−0.81
1MNM
34
V
0.58
1MNM
35
T
0.64
1MNM
36
F
4.05
1MNM 37 S 0.43
1MNM
38
K
4.05
1MNM 39
R
4.05
1MNM
40
K
2.83
1MNM
41
H
−1.24
1MNM
43
I
3.24
1MNM
45
K
4.05
1MNM 46 K
4.05
1MNM
48
F
−0.13
1MNM 49
E
2.27
1MNM
51
S
0
1MNM
52
V
0.11
1MNM 53 L 4.05
1MNM
66
T
0.64
1BDT 4
M
1.10 B own
e
al.
(1994)
1BDT
5
S
1.30
1BDT
6
K
1.30
1BDT
7
M
1.90
1BDT
9
Q
1.80
1BDT 11 N 2.00
1BDT
13
R
6.00
1BDT 23
R
0.20
1BDT
29
N
−1.00
1BDT
31
R
−0.30
1BDT
32
S
−2.30
1BDT
33
V
−2.30
1BDT 34 N 3.20
1BDT
35
S
−0.20
1BDT
39
Q
−0.40
1MSE
116
S
0.06 Oda
e
al.
(1997,
1998,
1999)
1MSE
139
N
0.60
1MSE 141
E
−0.10
1MSE
187
S
0.10
1B3T
469
R
3.41 C uickshank
e
al.
(2000)
1B3T
518
Y
2.62
1B3T
522
R
4.40
1QRV
9
L
0.02 Klass
e
al.
(2003)
1QRV
13
M
1.20
1QRV
32
V
−0.30
1J5N
18
P
0.00 Allain
e
al.
(1999)
1J5N
22
K
0.43
1J5N
28
Y
0.90
1J5N
33
N
0.74
1J5N
36
R
0.84
1J5N
40
R
0.72
1J5N
53
K
0.49
1J5N
54
K
0.00
1J5N
58
K
0.00
1J5N
60
K
0.40
1J5N
67
K
0.25
1J5N
78
K
0.43
1J5N 81
Y
0.21
1J5N
85
K
0.21
1J5N 88
Y
0.36
1JMC 234
R
2.15 Wal he
e
al.
(1999)
and
Wyka
e
al.
(2003)
1JMC
238
F
0.24
Table
2
(Con inued)
P o ein
ID
#AA
PDB
#AA
mu a ed
Gbinding/
kcal
mol−1
Re e ence
1JMC
263
K
1.01
1JMC 277 E
1.36
1JMC 382
R
1.91
1QZH
62
T
1.53
1QZH
64
D
1.46
1QZH
88
F
3.96
1QZH
91
Q
1.26
1QZH 115 Y 0.66
1QZH 122 L
1.00
1QZH 123
S
−0.70
1TN9
5
R
0.78 Connolly
e
al.
(2000)
1TN9
15
T
0.08
1TN9
18
S
−0.14
1TN9 21 K 0.74
1TN9
24
R
1.25
1TN9 26
L
−0.19
1TN9
42
W
0.48
1TN9 54 K 1.37
2A0I
3
S
4.15 La kin
e
al.
(2005)
2A0I 8
R
1.70
2A0I
19
D
−0.30
2A0I
88
K
5.57
2A0I
147
D
0.30
2A0I
148
T
0.30
2A0I 149 S 3.41
2A0I
150
R
2.92
2A0I 153
E
1.70
2A0I
155
Q
2.20
2A0I
158
T
0.90
2A0I 187 E 2.10
2A0I
220
K
0.80
2A0I 221 H
2.82
2A0I
223
M
2.70
2A0I 237
R
4.39
2A0I
241
I
3.91
2A0I
242
R
1.20
2A0I 254 R
3.17
2A0I
265
K
1.80
3.
Resul s
The
X- ay
c ys allog aphic
s uc u es
o
complexes
a ailable
a
he
RCSB
P o ein
Da a
Bank,
as
well
as
he
idimensional
s uc-
u es
ha
a e
he
basis
o
he
ini ial
knowledge
o
his
wo k
a e
s a ic
and
do
no
show
he
con o ma ional
changes
ha
occu
in
he
sys em
o e
ime.
The
co ec
comp ehension
o
he
phenomena
ha
occu s
wi hin
he
p o ein–DNA
in e ace
such
as
he
s uc-
u al
adap abili y
and
he
igh
binding
mode
can
benefi
g ea ly
om
he
use
o
compu a ional
me hods
capable
o
gene a ing
in o -
ma ion
based,
no
on
a
single
s uc u e,
bu
on
an
ensemble
o
con o ma ions
gene a ed
by
MD
simula ions.
I
also
makes
possible
o
unde s and
he
s uc u al
and
unc ional
ole
o
wa e
a ound
HS
and
NS,
since
he
g ea
majo i y
o
biological
p ocesses
occu
in
an
aqueous
medium.
Ou
da ase
consis s
o
10
p o ein–DNA
complexes
ha
we e
p e iously
desc ibed
in
he
me hodological
sec ion
and
a
o al
o
112
in e acial
esidues
(Table
2).
They
ha e
he
ollowing
dis ibu ion
by
amino
acid,
g oup
ype
and
hei
ho
and
null-spo
cha ac e :
Glu
(7%),
Phe
(4%),
His
(3%),
Ile
(4%),
Lys
(20%),
Leu
(4%),
Me
(4%),
Asn
(5%),
Gln
(4%),
A g
(17%),
Se
(10%),
Th
(5%),
Val
(4%)
and
Ty
(4%);
50%
a e
cha ged
esidues,
29%
pola
and
21%
nonpola ;
28%
a e
HS
and
72%
NS.
We
ha e
a
p edominance
o
cha ged
esidues,
especially
posi i ely
cha ged
amino
acids
such
as
Lys
and
A g,
which
is
consis en
wi h
p e ious
s udies
wi hin
p o ein–DNA
in e aces
ha
cha ac e ize
hem
as
highly
cha ged
in e aces
(Ahmad
e
al.,
2008).
The
measu e
o
he
RDF
p ofile
o
esidues
is
a
common
p o-
cedu e
when
dealing
wi h
a
sys em
o med
wi h
explici
sol en ,
since
i
allows
he
cha ac e iza ion
o
he
in e ac ion
be ween
he

R.M.
Ramos
e
al.
/
Compu a ional
Biology
and
Chemis y
44
(2013)
31–39
35
Fig.
2.
Rep esen a ion
o
he
RDF
p ofile
and
a e age
numbe
o
wa e s
a ound
he
a e age
HS
(a,
c)
and
he
a e age
NS
(b,
d).
Table
3
A e age
numbe
o
wa e
molecules
a
dis ances
be ween
3
and
6˚
A
o
HS
and
NS,
o
each
o
he
s udied
complexes.
Complex
1MNM
1BDT
1MSE
1B3T
1QRV
1JN5
1JMC
1QZH
1TN9
2A0I
Dis ance/Å
HS
NS
HS
NS
HS
NS
HS
NS
HS
NS
HS
NS
HS
NS
HS
NS
HS
NS
HS
NS
3 1.00 1.43
0.84
0.89
– 1.79
0.21
–
–
0.01
–
1.50
0.68
0.29
0.00
0.43
–
0.70
0.53
1.89
4
2.97
4.21
2.23
3.12
–
4.23
1.87
–
–
1.28
–
4.35
2.90
0.78
0.08
1.63
–
2.30
1.65
4.17
5
6.14
9.62
3.66
6.50
–
8.97
3.98
–
–
3.24
–
9.07
4.63
1.45
0.98
3.70
–
4.69
3.75
8.63
6
10.76
17.15
5.90
11.22
–
14.76
5.93
–
–
6.16
–
15.75
7.14
2.55
1.88
7.60
–
8.46
6.54
14.88
solu e
and
he
sol en
molecules.
Usually,
a
wa e
RDF
p ofile
exhibi s
an
oscilla o y
p ofile
and
a
peak
due
o
he
p esence
o
hyd ogen
bonds.
We
measu ed
he
RDFs
o
he
112
esidues
in
ou
da ase .
Fig.
2
shows
wo
di e en
RDF
p ofiles
ha
ep esen
a
HS
and
a
NS.
As
i
can
be
easily
pe cei ed,
he
exhibi
beha io
is
qui e
di e en
be ween
he
wo.
In
Fig.
2b
he
peak
loca ed
abou
3˚
A
is
due
o
he
s ong
in e ac ion
be ween
he
hyd ogen
a oms
o
wa e
and
he
oxygen
a oms
o
he
ca bonyl
g oup
o
he
NS.
Some
o he
peaks
can
be
pe cei ed
in
he
emaining
plo ,
which
a e
less
defined,
due
o
he
in e ac ion
be ween
he
wa e
molecules
and
he
a oms
o
he
amino
acid
esidue,
wi h
he
excep ion
o
hyd ogen.
On
he
o he
hand,
as
can
be
seen
in
plo
(a)
o
Fig.
2,
he
peaks
a e
less
defined
when
we
a e
dealing
wi h
a
pu a i e
HS.
Plo s
(c)
and
(d)
show
he
a e age
numbe
o
wa e
molecules
a ound
he
HS
and
he
NS,
which
a e
clea ly
dis inc .
We
also
mea-
su ed
he
a e age
numbe
o
wa e
molecules
a ound
each
esidue,
using
dis ance
cu o
alues
o
3,
4,
5
and
6˚
A.
Table
3
summa izes
he
esul s
o
each
o
he
complexes
unde
s udy
and
Table
4
he
global
esul s
in
e ms
o
HS
and
NS.
These
esul s
we e
plo ed
in
a
g aphic
and
shown
in
Fig.
3
o
a
simple
analysis.
I
is
pe -
cep ible
ha
he
a e age
numbe
o
wa e
molecules
a ound
HS
is
no o iously
lowe
when
compa ed
o
NS.
This
di e ence
o
a
cu o
alue
o
3˚
A
is
no
significan ,
bu
as
we
inc ease
he
cu o
alue
o
4,
5
and
6˚
A
he
di e ence
inc eases.
As
some
indi idual
sys ems
do
no
possess
bo h
HS
and
NS
(due
o
he
di ficul ies
in
finding
expe imen al
binding
ee
ene gy
alues
upon
alanine
mu a ion
Table
4
A e age
numbe
o
wa e
molecules
a
dis ances
be ween
3
and
6˚
A
o
he
o al
o
HS
and
NS
o
all
he
complexes.
Dis ance/Å
HS
NS
3 0.70
1.13
4 2.23
3.42
5
4.63
7.24
6 7.92
12.64
o
hese
p o ein–DNA
complexes)
we
also
p esen
global
esul s
o
HS
and
NS,
which
allows
a
be e
unde s anding
o
he
phe-
nomenon
o
occlusion.
Fo
a
NS,
and
a
a
5˚
A
dis ance,
he e
a e
an
a e age
o
7
wa e
molecules
a ound
i ;
ins ead
o
an
HS,
he
a e -
age
numbe
o
wa e
molecules
dec eases
o
5.
The
a e age
numbe
o
wa e
molecules
a ound
a
HS
and
a
NS
a ies
om
0.70
o
7.92
and
1.13
o
12.64,
espec i ely.
A
sho e
dis ances
bo h
HS
and
NS
p esen
an
a e age
numbe
o
wa e
molecules
ela i ely
small,
wi h
less
han
2
wa e
molecules,
which
g adually
inc eases
wi h
he
dis ance.
As
we
ha e
p e iously
s a ed,
he
in e ace
is
gene -
ally
dis inguished
in
a
co e
and
a
im,
and
he
HS
a e
usually
a
he
co e.
Wi h
his
in
mind,
and
o
e e y
HS
in
ou
da ase ,
we
selec ed
he
esidues
wi hin
4˚
A
and
be ween
4
and
8˚
A
dis ances
o
ou
HS
and
calcula ed
he
a e age
numbe
o
wa e
molecules
o
each
o
hese
coo dina ion
sphe es.
In
heo y
his
would
gi e
us
an
idea
o
Fig.
3.
Rep esen a ion
o
he
global
a e age
esul s
o
wa e
molecules
a ound
HS
and
NS.
36
R.M.
Ramos
e
al.
/
Compu a ional
Biology
and
Chemis y
44
(2013)
31–39
Fig.
4.
Rep esen a ion
o
he
a e age
numbe
o
wa e s
a ound
he
HS
(in
b own),
he
4˚
A
sol a ion
sphe e
(o ange);
and
he
8˚
A
sol a ion
sphe e
(g een).
(Fo
in e -
p e a ion
o
he
e e ences
o
colo
in
his
figu e
legend
and
he
ex ,
he
eade
is
e e ed
o
he
web
e sion
o
his
a icle.)
he
en i onmen
su ounding
each
HS
and
ac
as
a
complemen
o
he
co e
and
im
defini ion
o
he
in e ace.
The
esul s
ob ained
a e
shown
in
Fig.
4.
This
figu e
lis s
o
all
he
31
HS,
he
a e age
numbe
o
wa e s
o
he
HS
i sel
(b own),
o
he
esidues
inside
he
fi s
coo dina ion
sphe e
(o ange)
and
o
he
esidues
inside
he
second
coo dina ion
sphe e
(g een).
I
should
be
no ed
ha
in
his
figu e
he
s acked
e ical
ba s
o e
o
each
HS
he
gi en
pe cen age
o e
he
h ee
analyzed
da a
(HS,
4˚
A
sphe e
and
be ween
4
and
8˚
A
sphe e).
As
expec ed,
o
he
majo i y
o
he
analyzed
HS
he
a e -
age
numbe
o
wa e
molecules
wi hin
he
fi s
coo dina ion
sphe e
is
lowe
han
in
he
second.
The
esidues
ha
closely
su ound
he
HS,
and
belong
o
he
co e,
a e
mo e
bu ied
and
ha e
less
accessibil-
i y
o
he
sol en .
On
he
o he
hand
when
we
ad ance
o
he
nex
coo dina ion
sphe e,
which
can
be
ela ed
o
he
im
egion,
we
find
mo e
esidues
wi h
highe
sol en
accessibili y
ha
accoun
o
he
di e ence
ob ained.
Ne e heless,
some
o
he
esidues
wi hin
he
wo
sol a ion
sphe es
seem
o
p esen
a
di e en
beha io .
We
can-
no
exclude
ha
may
be
influenced
by
he
exis ence
o
a
HS
wi hin
hose
sphe es.
As
men ioned
in
he
me hodological
pa ,
he
known
HS
we e
excluded
bu
Gbinding alues
we e
no
a ailable
o
all
he
esidues
o
he
analyzed
in e aces.
The
esul s
we
ob ained,
based
on
MD
simula ions
o
p o ein–DNA
sys ems,
clea ly
show
ha
he
a e age
numbe
o
wa e
molecules
a ound
a
HS
is
much
lowe
when
compa ed
o
NS.
This
is
in
ag eemen
wi h
he
O- ing
heo y
and
he
ac
ha
he
HS
a e
gene ally
p o ec ed
om
he
sol en .
We
also
measu ed
di e en
SASA
ea u es
o
he
complex
and
he
monome s
o
com-
plemen
he
s udy
as
i
allows
a
mo e
p o ound
cha ac e iza ion
o
he
impo ance
o
he
wa e
molecules
in
he
mic o
ambien
o
he
HS
and
NS.
SASA
and
elSASA,
ha
we e
p e iously
desc ibed,
we e
also
measu ed.
The
esul s
we
ob ained
a e
summa ized
in
Table
5
and
Fig.
5.
Ou
esul s
ollow
he
endency
obse ed
o
PPI
and
indica e
ha
a
high
alue
o
SASA
and
elSASA
is
a
necessa y
condi ion
o
a
esidue
o
be
conside ed
a
HS.
Howe e ,
hese
ea u es
a e
insu ficien
o
make
a
clea
dis inc ion
be ween
HS
and
NS,
as
some
NS
also
p esen
a
high
alue
o
SASA.
The
ype
o
amino
acid
plays
a
c ucial
ole
in
he
defini ion
o
he
in e ace,
so
we
ha e
also
op ed
o
analyze
he
esul s
acco ding
o
he
ype
o
esidue:
cha ged
(Asp,
Glu,
His,
Lys
e
A g),
pola
(Se ,
Th ,
Asn,
Gln
e
Ty ),
nonpola
(Val,
Ile,
Leu,
Me ,
Phe
e
T p)
and
a oma ic
(Phe,
T p,
Ty
e
His).
Fig.
5
illus a es
he
di e en
SASA
and
elSASA
alues
achie ed
o
HS
and
NS.
The
di e ence
be ween
he
wo
se s
o
esidues
is
no o ious,
and
o
all
he
112
esidues
analyzed
we
Fig.
5.
Schema ic
ep esen a ion
o
he
a e age
SASA
and
elSASA
alues
o
each
o
he
amino
acid
g oups
conside ed.
ha e
ob ained
alues
o
35.03
±
4.31 ˚
A2(SASA)
and
0.54
±
0.05
( elSASA)
o
he
HS
and
26.57
±
4.83 ˚
A2(SASA)
and
0.30
±
0.05
( elSASA)
o
NS.
An
excep ion
can
be
ound
o
he
nonpola
esidues,
which
con ibu ion
is
highe
o
he
NS.
This
s a is ical
esul
is
influenced
by
he
small
numbe
o
nonpola
esidues
ha
ac
as
HS,
when
compa ed
o
he
numbe
o
NS.
Howe e ,
i
should
be
no ed
ha
he
a e age
SASA
alue
o
he
nonpola
NS
(31.08
±
4.92 ˚
A2)
is
wi hin
he
expec ed
alue
o
a
ypical
HS,
which
indica es
ha
he
nonpola
esidues
ha e
low
accessibili y
o
sol en .
Cha ged,
pola
and
a oma ic
esidues
show
a
clea
di e ence
be ween
HS
and
NS,
wi h
HS
ha ing
highe
SASA
and
elSASA.
We
ha e
o
highligh
ha
o
cha ged,
a oma ic
and
pola
esidues
he
di e ence
be ween
HS
and
NS
is
significan ,
especially
o
elSASA
in
which
he
di e ence
be ween
HS
and
NS
o
cha ged
and
pola
esidues
is
mo e
han
he
double.
We
ook
a
simila
app oach
as
desc ibed
o
he
RDF
calcula ions.
We
ha e
also
measu ed
he
SASA
cha ac e is ics
o
he
esidues
inside
he
4˚
A
and
be ween
4and
8˚
A
sphe es
a ound
he
HS
esidues,
and
compa ed
he
esul s
wi h
he
HS
by
hemsel es.
The
esul s
we e
plo ed
in
Fig.
6
(SASA)
and
Fig.
7
( elSASA).
Bo h
plo s,
especially
Fig.
7,
show
a
simila
and
expec ed
end.
The
HS
ha e
he
highe
alue
o
elSASA
(o
SASA).
As
we
ad ance
o
he
fi s
coo dina ion
sphe e
mo e
esidues
a e
ound
wi h
highe
accessibili y
o
sol en ,
and
he e o e
he e
is
a
dec ease
in
elSASA.
I
is
mo e
no o ious
when
we
conside ed
he
second
coo dina ion
sphe e
ha
is
o med
almos
by
NS
and
esidues
wi h
high
R.M.
Ramos
e
al.
/
Compu a ional
Biology
and
Chemis y
44
(2013)
31–39
37
Table
5
A e age
and
s anda d
de ia ion
alues
o
SASA
and
elSASA,
o
each
o
he
conside ed
amino
acid
g oups.
Amino
acid
g oups
HS
NS
SASA/[Å2]All
35.03
±
4.31
26.57
±
4.83
ASP
+
GLU 13.08
±
4.66
5.50
±
2.06
LYS
+
ARG
+
HIS 41.37
±
5.04
32.89
±
7.13
Cha ged
38.04
±
4.99
26.57
±
5.96
Pola
37.54
±
3.94
23.79
±
3.09
A oma ic
27.80
±
3.19
24.45
±
4.91
Nonpola
18.00
±
2.07
31.08
±
4.92
elSASA All 0.54
±0.05
0.30
±
0.05
ASP
+
GLU 0.22
±
0.09
0.15
±
0.05
LYS
+
ARG
+
HIS
0.57
±
0.05
0.25
±
0.05
Cha ged
0.53
±
0.05
0.20
±
0.05
Pola
0.84
±
0.05
0.32
±
0.04
A oma ic
0.35
±
0.02
0.33
±
0.04
Nonpola
0.34
±
0.05
0.44
±
0.05
Fig.
6.
Schema ic
ep esen a ion
o
he
a e age
SASA
conside ing
he
wo
coo dina ion
sphe es
analyzed
o
all
HS.
Fig.
7.
Schema ic
ep esen a ion
o
he
a e age
elSASA
conside ing
he
wo
coo dina ion
sphe es
analyzed
o
all
HS.
sol en
accessibili y.
Globally,
we
ob ained
on
a e age
32.77
±
4.04 ˚
A2,
13.32
±
2.25 ˚
A2and
12.67
±
2.00 ˚
A2 o
SASA
on
he
HS
i sel ,
fi s
coo dina ion
sphe e
and
second
coo dina ion
sphe e,
espec i ely;
0.54
±
0.05,
0.28
±
0.04
and
0.21
±
0.03
o
elSASA
on
he
HS,
fi s
and
second
coo dina ion
sphe es,
espec-
i ely.
Toge he
wi h
he
RDF
calcula ions,
he
SASA
ea u es
seem
o
confi m
ha
he
O- ing
heo y
is
applicable
o
p o ein–DNA
in e aces.
Ne e heless,
we
ha e
o
s ess
ou
ha
ou
da ase
is
composed
o
en
complexes,
wi h
a
known
X- ay
s uc u e
ha
ulfills
he
condi ions
men ioned
in
he
me hodological
pa .
As
long
as
new
expe imen al
da a
becomes
a ailable
he
s udy
should
be
ex ended.
Enough
expe imen al
da a
could
also
allow
he
di e en ia ion
be ween
he
di e en
ypes
o
p o ein–DNA
in e ace:
enzyme,
ansc ip ion
ac o
and
suppo ing
p o eins.
38
R.M.
Ramos
e
al.
/
Compu a ional
Biology
and
Chemis y
44
(2013)
31–39
4.
Conclusions
In
he
las
yea s
he e
has
been
an
e o
o
unde s and
he
p o-
cesses
ha
go e n
he
o ma ion
o
biological
complexes
and
he
in e ac ions
ha
a e
behind
hem.
The
binding
o
p o eins
wi h
o he
p o eins,
ligands
o
nucleic
acids
cons i u es
one
o
hese
p ocesses
ha
ecen ly
ha e
been
compu a ional
s udied.
Fo
PPI
i
was
p oposed
ha
only
a
small
ac ion
o
esidues
con ibu e
significan ly
o
he
binding
ee
ene gy
–
he
ho -spo s
–
which
a e
p o ec ed
om
sol en
molecules
by
null-spo s.
This
heo y
la e
known
as
he
O- ing
heo y
also
s a es
ha
o
a
esidue
o
be
conside ed
a
HS
i
should
ha e
a
low
alue
o
SASA.
The
O- ing
heo y
has
been
he
cen al
aspec
o
a
numbe
o
scien ific
pape s,
bu
he
s udies
we e
mos ly
pe o med
in
PP
complexes,
and
i s
s udy
wi h
o he
ypes
o
in e aces
such
as
he
PDI
is
missing.
By
measu ing
he
a e age
SASA
ea u es
o
he
112
esidues
o
en
dis inc
p o ein–DNA
complexes
i
was
possible
o
ob ain
a
clea
pe spec i e
o
he
beha io
o
HS
and
NS.
Radial
dis ibu ion
unc-
ions
we e
also
measu ed
and
helped
o
clea ly
dis inguish
be ween
he
wo
ypes
o
esidues.
Ou
esul s
show
ha
he
HS
end
o
ha e
ewe
wa e
molecules
in
hei
mic o
ambien
and
a
highe
alue
o
SASA.
So,
hey
a e
occluded
om
he
sol en
by
he
NS,
which
ones
ha e
mo e
wa e
molecules
in
hei
neighbo hood.
In
his
wo k
we
we e
able
o
ex end
he
applicabili y
o
he
O- ing
heo y
o
p o ein–DNA
complexes,
since
i
was
ini ially
o -
mula ed
in
p o ein–p o ein
complexes.
We
p esen
e idence
ha
he
HS
a e
indeed
occluded
om
bulk
sol en .
Toge he
wi h
he
p e ious
wo ks
de eloped
in
p o ein–p o ein
in e aces,
i
ali-
da es
he
O- ing
heo y.
Re e ences
Ac on,
T.B.,
Mead,
J.,
S eine ,
A.M.,
Ve shon,
A.K.,
2000.
Scanning
mu agenesis
o
MCM1:
esidues
equi ed
o
DNA
binding,
DNA
bending,
and
ansc ip ional
ac i a ion
by
a
Mads-Box
p o ein.
Molecula
and
Cellula
Biology
20,
1–11.
Ahmad,
S.,
Keskin,
O.,
Sa ai,
A.,
Nussino ,
R.,
2008.
P o ein–DNA
in e ac ions:
s uc u al,
he modynamic
and
clus e ing
pa e ns
o
conse ed
esidues
in
DNA-binding
p o eins.
Nucleic
Acids
Resea ch
36,
5922–5932.
Allain,
F.H.T.,
Yen,
Y.M.,
Masse,
J.E.,
Schul ze,
P.,
Dieckmann,
T.,
Johnson,
R.C.,
Feigon,
J.,
1999.
Solu ion
s uc u e
o
he
HMG
p o ein
NHP6A
and
i s
in e ac ion
wi h
DNA
e eals
he
s uc u al
de e minan s
o
non-sequence-specific
binding.
Embo
Jou nal
18,
2563–2579.
Bahadu ,
R.P.,
Chak aba i,
P.,
Rodie ,
F.,
Janin,
J.,
2003.
Dissec ing
subuni
in e -
aces
in
homodime ic
p o eins.
P o eins-S uc u e
Func ion
and
Gene ics
53,
708–719.
Bas,
D.C.,
Roge s,
D.M.,
Jensen,
J.H.,
2008.
Ve y
as
p edic ion
and
a ionaliza ion
o
Pk(a)
alues
o
p o ein–ligand
complexes.
P o eins-S uc u e
Func ion
and
Bioin o ma ics
73,
765–783.
Bochka e ,
A.,
P ue zne ,
R.A.,
Edwa ds,
A.M.,
F appie ,
L.,
1997.
S uc u e
o
he
single-s anded-DNA-binding
domain
o
eplica ion
p o ein
a
bound
o
DNA.
Na u e
385,
176–181.
Bochka e ,
A.,
Bochka e a,
E.,
F appie ,
L.,
Edwa ds,
A.M.,
1998.
The
2.2
Angs om
s uc u e
o
a
pe mangana e-sensi i e
DNA
si e
bound
by
he
Eps ein-Ba
i us
o igin
binding
p o ein,
EBNA1.
Jou nal
o
Molecula
Biology
284,
1273–1278.
Bogan,
A.A.,
Tho n,
K.S.,
1998.
Ana omy
o
ho
spo s
in
p o ein
in e aces.
Jou nal
o
Molecula
Biology
280,
1–9.
B ooks,
B.R.,
B uccole i,
R.E.,
Ola son,
B.D.,
S a es,
D.J.,
Swamina han,
S.,
Ka plus,
M.,
1983.
Cha mm:
a
p og am
o
mac omolecula
ene gy,
minimiza ion,
and
dynamics
calcula ions.
Jou nal
o
Compu a ional
Chemis y
4,
187–217.
B own,
B.M.,
Milla,
M.E.,
Smi h,
T.L.,
Saue ,
R.T.,
1994.
Scanning
mu agenesis
o
he
a c
ep esso
as
a
unc ional
p obe
o
ope a o
ecogni ion.
Na u e
S uc u al
Biology
1,
164–168.
Case,
D.A.,
Da den,
T.A.,
Chea ham,
T.E.,
Simme ling,
C.L.,
Wang,
J.,
Duke,
R.E.,
Luo,
R.,
Me z,
K.M.,
Pea lman,
D.A.,
C owley,
M.,
Walke ,
R.C.,
Zhang,
W.,
Wang,
B.,
Hayik,
S.,
Roi be g,
A.,
Seab a,
G.,
Wong,
K.F.,
Paesani,
F.,
Wu,
X.,
B ozell,
S.,
Tsui,
V.,
Gohlke,
H.,
Yang,
L.,
Tan,
C.,
Mongan,
J.,
Ho nak,
V.,
Cui,
G.,
Be oza,
P.,
Ma hews,
D.H.,
Scha meis e ,
C.,
Ross,
W.S.,
Kollman,
P.A.,
2006.
Ambe
9.
Uni e si y
o
Cali o nia,
San
F ancisco.
Chak aba i,
P.,
Janin,
J.,
2002.
Dissec ing
p o ein–p o ein
ecogni ion
si es.
P o eins-
S uc u e
Func ion
and
Gene ics
47,
334–343.
Cho hia,
C.,
Janin,
J.,
1975.
P inciples
o
p o ein–p o ein
ecogni ion.
Na u e
256,
705–708.
Clackson,
T.,
Ul sch,
M.H.,
Wells,
J.A.,
de
Vos,
A.M.,
1998.
S uc u al
and
unc ional
analysis
o
he
1:
1
g ow h
ho mone:
ecep o
complex
e eals
he
molecula
basis
o
ecep o
a fini y.
Jou nal
o
Molecula
Biology
277,
1111–1128.
Connolly,
K.M.,
Ilango an,
U.,
Wojciak,
J.M.,
Iwaha a,
M.,
Clubb,
R.T.,
2000.
Majo
g oo e
ecogni ion
by
h ee-s anded
be a-shee s:
a fini y
de e minan s
and
conse ed
s uc u al
ea u es.
Jou nal
o
Molecula
Biology
300,
841–856.
C uickshank,
J.,
Shi e,
K.,
Da idson,
A.R.,
Edwa ds,
A.M.,
F appie ,
L.,
2000.
Two
domains
o
he
Eps ein-Ba
i us
o igin
DNA-binding
p o ein,
EBNA1,
o ches-
a e
sequence-specific
DNA
binding.
Jou nal
o
Biological
Chemis y
275,
22273–22277.
Da den,
T.,
Yo k,
D.,
Pede sen,
L.,
1993.
Pa icle
mesh
Ewald
–an
N·Log(N)
me hod
o
Ewald
sums
in
la ge
sys ems.
Jou nal
o
Chemical
Physics
98,
10089–10092.
DeLano,
W.L.,
2002.
Un a eling
ho
spo s
in
binding
in e aces:
p og ess
and
chal-
lenges.
Cu en
Opinion
in
S uc u al
Biology
12,
14–20.
DeLano,
W.L.,
Ul sch,
M.H.,
de
Vos,
A.M.,
Wells,
J.A.,
2000.
Con e gen
solu ions
o
binding
a
a
p o ein–p o ein
in e ace.
Science
287,
1279–1283.
Guha oy,
M.,
Chak aba i,
P.,
2005.
Conse a ion
and
ela i e
impo ance
o
esidues
ac oss
p o ein–p o ein
in e aces.
P oceedings
o
he
Na ional
Academy
o
Sci-
ences
o
he
Uni ed
S a es
o
Ame ica
102,
15447–15452.
Ho nak,
V.,
Abel,
R.,
Oku ,
A.,
S ockbine,
B.,
Roi be g,
A.,
Simme ling,
C.,
2006.
Compa ison
o
mul iple
ambe
o ce
fields
and
de elopmen
o
imp o ed
p o-
ein
backbone
pa ame e s.
P o eins:
S uc u e,
Func ion,
and
Bioin o ma ics
65,
712–725.
Humph ey,
W.,
Dalke,
A.,
Schul en,
K.,
1996.
VMD:
isual
molecula
dynamics.
Jou -
nal
o
Molecula
G aphics
14,
33–38.
Huo,
S.,
Masso a,
I.,
Kollman,
P.A.,
2002.
Compu a ional
alanine
scanning
o
he
1:
1
human
g ow h
ho mone- ecep o
complex.
Jou nal
o
Compu a ional
Chemis y
23,
15–27.
Izagui e,
J.A.,
Ca a ello,
D.P.,
Wozniak,
J.M.,
Skeel,
R.D.,
2001.
Lange in
s abiliza ion
o
molecula
dynamics.
Jou nal
o
Chemical
Physics
114,
2090–2098.
Janin,
J.,
1995.
Elusi e
a fini ies.
P o eins-S uc u e
Func ion
and
Gene ics
21,
30–39.
Jaya am,
B.,
McConnell,
K.,
Dixi ,
S.B.,
Das,
A.,
Be e idge,
D.L.,
2002.
F ee-ene gy
componen
analysis
o
40
p o ein–DNA
complexes:
a
consensus
iew
on
he
he modynamics
o
binding
a
he
molecula
le el.
Jou nal
o
Compu a ional
Chemis y
23,
1–14.
Jones,
S.,
Tho n on,
J.M.,
1996.
P inciples
o
p o ein–p o ein
in e ac ions.
P oceedings
o
he
Na ional
Academy
o
Sciences
o
he
Uni ed
S a es
o
Ame ica
93,
13–20.
Jo gensen,
W.L.,
Chand asekha ,
J.,
Madu a,
J.D.,
Impey,
R.W.,
Klein,
M.L.,
1983.
Com-
pa ison
o
simple
po en ial
unc ions
o
simula ing
liquid
wa e .
Jou nal
o
Chemical
Physics
79,
926–935.
Klass,
J.,
Mu phy,
F.V.,
Fou s,
S.,
Se enil,
M.,
Changela,
A.,
Siple,
J.,
Chu chill,
M.E.A.,
2003.
The
ole
o
in e cala ing
esidues
in
ch omosomal
high-mobili y-g oup
p o ein
DNA
binding,
bending
and
specifici y.
Nucleic
Acids
Resea ch
31,
2852–2864.
Koslo ,
M.,
T a is,
A.M.,
Bosch,
D.E.,
Side o ski,
D.P.,
A sha sky,
V.Y.,
2011.
In e-
g a ing
ene gy
calcula ions
wi h
unc ional
assays
o
deciphe
he
specifici y
o
G
p o ein–RGS
p o ein
in e ac ions.
Na u e
S uc u al
&
Molecula
Biology
18,
846-U128.
Kuma ,
M.D.S.,
Ba a,
K.A.,
G omiha,
M.M.,
P abaka an,
P.,
Ki ajima,
K.,
Uedai a,
H.,
Sa ai,
A.,
2006.
PROTHERM
and
PRONIT:
he modynamic
da abases
o
p o-
eins
and
p o ein–nucleic
acid
in e ac ions.
Nucleic
Acids
Resea ch
34,
D204–
D206.
La kin,
C.,
Da a,
S.,
Ha ley,
M.J.,
Ande son,
B.J.,
Ebie,
A.,
Ha g ea es,
V.,
Schild-
bach,
J.F.,
2005.
In e -
and
in amolecula
de e minan s,
o
he
specifici y
o
single-s anded
DNA
binding
and
clea age
by
he
F
ac o
elaxase.
S uc u e
13,
1533–1544.
Lee,
B.,
Richa ds,
F.M.,
1971.
The
in e p e a ion
o
p o ein
s uc u es:
es ima ion
o
s a ic
accessibili y.
Jou nal
o
Molecula
Biology
55,
379–400.
Lei,
M.,
Podell,
E.R.,
Baumann,
P.,
Cech,
T.R.,
2003.
DNA
Sel - ecogni ion
in
he
s uc-
u e
o
POT1
bound
o
elome ic
single-s anded
DNA.
Na u e
426,
198–203.
Li,
J.,
Liu,
Q.,
2009.
‘Double
Wa e
Exclusion’:
a
hypo hesis
efining
he
O- ing
heo y
o
he
ho
spo s
a
p o ein
in e aces.
Bioin o ma ics
25,
743–750.
Li,
H.,
Robe son,
A.D.,
Jensen,
J.H.,
2005.
Ve y
as
empi ical
p edic ion
and
a ional-
iza ion
o
p o ein
Pk(a)
alues.
P o eins-S uc u e
Func ion
and
Bioin o ma ics
61,
704–721.
Loncha ich,
R.J.,
B ooks,
B.R.,
Pas o ,
R.W.,
1992.
Lange in
dynamics
o
pep ides
– he
ic ional
dependence
o
isome iza ion
a es
o
N-ace ylalanyl-N-me hylamide.
Biopolyme s
32,
523–535.
Masse,
J.E.,
Wong,
B.,
Yen,
Y.M.,
Allain,
F.H.T.,
Johnson,
R.C.,
Feigon,
J.,
2002.
The
S.
Ce e isiae
a chi ec u al
HMGB
P o ein
NHP6A
complexed
wi h
DNA:
DNA
and
p o ein
con o ma ional
changes
upon
binding.
Jou nal
o
Molecula
Biology
323,
263–284.
Masso a,
I.,
Kollman,
P.A.,
1999.
Compu a ional
alanine
scanning
o
p obe
p o ein–p o ein
in e ac ions:
a
no el
app oach
o
e alua e
binding
ee
ene gies.
Jou nal
o
he
Ame ican
Chemical
Socie y
121,
8133–8143.
Mo ei a,
I.S.,
Fe nandes,
P.A.,
Ramos,
M.J.,
2006a.
De ailed
mic oscopic
s udy
o
he
ull
ZIPA:FTSZ
in e ace.
P o eins-S uc u e
Func ion
and
Bioin o ma ics
63,
811–821.
Mo ei a,
I.S.,
Fe nandes,
P.A.,
Ramos,
M.J.,
2006b.
Un a eling
he
impo ance
o
p o ein–p o ein
in e ac ion:
applica ion
o
a
compu a ional
alanine-scanning
mu agenesis
o
he
s udy
o
he
IGG1
s ep ococcal
p o ein
G
(C2
agmen )
complex.
Jou nal
o
Physical
Chemis y
B
110,
10962–10969.
Mo ei a,
I.S.,
Fe nandes,
P.A.,
Ramos,
M.J.,
2007a.
Compu a ional
alanine
scanning
mu agenesis
–
an
imp o ed
me hodological
app oach.
Jou nal
o
Compu a ional
Chemis y
28,
644–654.
Mo ei a,
I.S.,
Fe nandes,
P.A.,
Ramos,
M.J.,
2007b.
Ho
spo
occlusion
om
bulk
wa e :
a
comp ehensi e
s udy
o
he
complex
be ween
he
Lysozyme
Hel
and
he
An i-
body
FVD1.3.
Jou nal
o
Physical
Chemis y
B
111,
2697–2706.
R.M.
Ramos
e
al.
/
Compu a ional
Biology
and
Chemis y
44
(2013)
31–39
39
Mo ei a,
I.S.,
Fe nandes,
P.A.,
Ramos,
M.J.,
2007c.
Ho
spo s
–
a
e iew
o
he
p o ein–p o ein
in e ace
de e minan
amino-acid
esidues.
P o eins-S uc u e
Func ion
and
Bioin o ma ics
68,
803–812.
Mu phy,
F.V.,
Swee ,
R.M.,
Chu chill,
M.E.A.,
1999.
The
s uc u e
o
a
ch omosomal
high
mobili y
g oup
p o ein–DNA
complex
e eals
sequence-neu al
mecha-
nisms
impo an
o
non-sequence-specific
DNA
ecogni ion.
Embo
Jou nal
18,
6610–6618.
Oda,
M.,
Fu ukawa,
K.,
Oga a,
K.,
Sa ai,
A.,
Ishii,
S.,
Nishimu a,
Y.,
Nakamu a,
H.,
1997.
Iden ifica ion
o
indispensable
esidues
o
specific
DNA-binding
in
he
impe ec
andem
epea s
o
C-Myb
R2 3.
P o ein
Enginee ing
10,
1407–1414.
Oda,
M.,
Fu ukawa,
K.,
Oga a,
K.,
Sa ai,
A.,
Nakamu a,
H.,
1998.
The modynamics
o
specific
and
non-specific
DNA
binding
by
he
C-Myb
DNA-binding
domain.
Jou nal
o
Molecula
Biology
276,
571–590.
Oda,
M.,
Fu ukawa,
K.,
Sa ai,
A.,
Nakamu a,
H.,
1999.
Kine ic
analysis
o
DNA
bind-
ing
by
he
C-Myb
DNA-binding
domain
using
su ace
plasmon
esonance.
FEBS
Le e s
454,
288–292.
Oga a,
K.,
Mo ikawa,
S.,
Nakamu a,
H.,
Sekikawa,
A.,
Inoue,
T.,
Kanai,
H.,
Sa ai,
A.,
Ishii,
S.,
Nishimu a,
Y.,
1994.
Solu ion
s uc u e
o
a
specific
DNA
complex
o
he
Myb
DNA-binding
domain
wi h
coope a i e
ecogni ion
helices.
Cell
79,
639–
648.
Olsson,
M.H.M.,
Sonde gaa d,
C.R.,
Ros kowski,
M.,
Jensen,
J.H.,
2011.
PROPKA3:
con-
sis en
ea men
o
in e nal
and
su ace
esidues
in
empi ical
Pk(a)
p edic ions.
Jou nal
o
Chemical
Theo y
and
Compu a ion
7,
525–537.
Pé ez,
A.,
Luque,
F.J.,
O ozco,
M.,
2011.
F on ie s
in
molecula
dynamics
simula ions
o
DNA.
Accoun s
o
Chemical
Resea ch
45,
196–205.
P abaka an,
P.,
An,
J.,
G omiha,
M.M.,
Sel a aj,
S.,
Uedai a,
H.,
Kono,
H.,
Sa ai,
A.,
2001.
The modynamic
da abase
o
p o ein–nucleic
acid
in e ac ions
(P oni ).
Bioin o ma ics
17,
1027–1034.
Rajamani,
D.,
Thiel,
S.,
Vajda,
S.,
Camacho,
C.J.,
2004.
Ancho
esidues
in
p o ein–p o ein
in e ac ions.
P oceedings
o
he
Na ional
Academy
o
Sciences
o
he
Uni ed
S a es
o
Ame ica
101,
11287–11292.
Ryckae ,
J.P.,
Cicco i,
G.,
Be endsen,
H.J.C.,
1977.
Nume ical-in eg a ion
o
ca esian
equa ions
o
mo ion
o
a
sys em
wi h
cons ain s
– molecula -dynamics
o
N-
alkanes.
Jou nal
o
Compu a ional
Physics
23,
327–341.
Schildbach,
J.F.,
Ka zai,
A.W.,
Raumann,
B.E.,
Saue ,
R.T.,
1999.
O igins
o
DNA-binding
specifici y:
ole
o
p o ein
con ac s
wi h
he
DNA
backbone.
P oceedings
o
he
Na ional
Academy
o
Sciences
o
he
Uni ed
S a es
o
Ame ica
96,
811–817.
Tan,
S.,
Richmond,
T.J.,
1998.
C ys al
s uc u e
o
he
yeas
ma
alpha
2/MCM1/DNA
e na y
complex.
Na u e
391,
660–666.
Tho n,
K.S.,
Bogan,
A.A.,
2001.
ASEDB:
a
da abase
o
alanine
mu a ions
and
hei
e ec s
on
he
ee
ene gy
o
binding
in
p o ein
in e ac ions.
Bioin o ma ics
17,
284–285.
Wal he ,
A.P.,
Gomes,
X.V.,
Lao,
Y.,
Lee,
C.G.,
Wold,
M.S.,
1999.
Replica ion
p o ein
a
in e ac ions
wi h
DNA,
1.
Func ions
o
he
DNA-binding
and
zinc-finge
domains
o
he
70-Kda
subuni .
Biochemis y
38,
3963–3973.
Wojciak,
J.M.,
Connolly,
K.M.,
Clubb,
R.T.,
1999.
Nm
s uc u e
o
he
TN916
in eg ase-DNA
complex.
Na u e
S uc u al
Biology
6,
366–373.
Wyka,
I.M.,
Dha ,
K.,
Binz,
S.K.,
Wold,
M.S.,
2003.
Replica ion
p o ein
a
in e ac ions
wi h
DNA:
di e en ial
binding
o
he
co e
domains
and
analysis
o
he
DNA
in e ac ion
su ace.
Biochemis y
42,
12909–12918.