Study of Protein-nucleic acid Complexes

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. 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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. 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