Molecules 2014, 19, 12531-12546; doi:10.3390/molecules190812531
molecules
ISSN 1420-3049
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A icle
pH-Dependen De o ma ions o he Ene gy Landscape o
A idin-like P o eins In es iga ed by Single Molecule
Fo ce Spec oscopy
Melanie Köhle 1, And eas Ka ne 2, Michael Lei ne 2, Vesa P. Hy önen 3,4, Ma kku Kulomaa 3,
Pe e Hin e do e 1,2 and And eas Ebne 1,*
1 Ins i u e o Biophysics, Johannes Keple Uni e si y Linz, G ube s asse 40, 4020 Linz, Aus ia;
E-Mails: melanie.koehle @jku.a (M.K.); pe e .hin [email p o ec ed] (P.H.)
2 Cen e o Ad anced Bioanalysis, G ube s asse 40, 4020 Linz, Aus ia;
E-Mails: [email p o ec ed] (A.K.); michael.lei ne @cbl.a (M.L.)
3 Ins i u e o Biomedical Technology, Uni e si y o Tampe e, FI-33014 Tampe e, Finland;
E-Mails: [email p o ec ed] (V.P.H.), m[email p o ec ed] (M.K.)
4 Fimlab Labo a o ies L d., FI-33101 Tampe e, Finland
*Au ho o whom co espondence should be add essed; E-Mail: a[email p o ec ed];
Tel.: +43-732-2468-7637; Fax: +43-732-2468-7633.
Recei ed: 16 June 2014; in e ised o m: 31 July 2014 / Accep ed: 7 Augus 2014 /
Published: 18 Augus 2014
Abs ac : A idin and a idin-like p o eins a e widely used in nume ous echniques since
he a idin-bio in in e ac ion is known o be e y obus and eliable. Wi hin his s udy, we
in es iga ed his bond a he molecula le el unde ha sh condi ions anging om e y low
o e y high pH alues. We compa ed a idin wi h s ep a idin and a ecen ly de eloped
a idin-based mu an , chime ic a idin. To gain insigh s o he ene gy landscape o hese
in e ac ions we used a single molecule app oach and pe o med he Single Molecule Fo ce
Spec oscopy a omic o ce mic oscopy echnique. The e, he ligand (bio in) is co alen ly
coupled o a sha p AFM ip ia a dis ensible he e o-bi- unc ional c osslinke , whe eas he
ecep o o in e es is immobilized on he p obe su ace. Recep o -ligand complexes a e
o med and up u ed by epea edly app oaching and wi hd awing he ip om he su ace.
Va ying bo h pulling eloci y and pH alue, we could de e mine changes o he ene gy
landscape o he complexes. Ou esul s clea ly demons a e ha a idin, s ep a idin and
chime ic a idin a e s able o e a wide pH ange al hough we could iden i y di e ences a
he ou e pH ange. Taking his in o accoun , hey can be used in a b oad ange o
applica ions, like su ace senso s a ex eme pH alues.
OPEN ACCESS
Molecules 2014, 19 12532
Keywo ds: a idin mu an ; a idin-bio in; o ce spec oscopy; molecula ecogni ion;
pH dependence; single molecules; biophysics
1. In oduc ion
A idin and a idin-like p o eins anging om a idin and s ep a idin o mo e ecen ly de eloped
mu an s like chime ic a idin and ap a idin [1–3] a e known o play a majo ole in li e-science
applica ions ( o e iews see [4–6]). A idin-like p o eins a e commonly buil up as a homo e ame ,
whe eby each monome has a single eigh -s anded β-ba el ac ing as binding pocke o D-bio in. The
wide ange o biochemical, biophysical, pha maceu ical, and medical applica ions is a logical esul o
nume ous ad an ages: (i) a idin (and mos o he a idin-like p o eins) shows an ex emely high
a ini y owa ds he wa e -soluble i amin D-bio in (Kd ~10−15 M). D-Bio in i sel is a e y small
molecule ha can be easily bound co alen ly o p o eins wi h a low p obabili y o ha m hei unc ional
p ope ies. (ii) The sys em is a ailable o a b oad ange o applica ions and compa able cheap. In
addi ion (iii) a idin/bio in is known o be e y obus and eliable. In p e ious s udies he capabili y o
(s ep )a idin (mu an s) o bind bio in a e p e ea men a di e en en i onmen al condi ions anging
om de e gen s [7] and o ganic sol en s, empe a u e changes [2,8], up o ha sh pH condi ions [2] was
in es iga ed. In all hese s udies an ex ao dina y s abili y o hese p o eins could be demons a ed.
Wi hin his s udy we explo ed he bio in binding beha io a ex eme condi ions o h ee p o eins:
a idin, s ep a idin and he a idin mu an chime ic a idin. Chime ic a idin is an a idin based mu an
known o ha e an inc eased he mal and pH s abili y and a be e esis ance agains he p o eoly ic
ac i i y o p o einase K. Chime ic a idin is p epa ed by eplacing a segmen in a idin wi h
co esponding sequence s e ch om a idin ela ed p o ein 4. In addi ion, he isoleucine a posi ion
117 is exchanged by y osine, mos p obably esul ing in a s abiliza ion e ec due o π-π in e ac ion o
wo y osines om adjacen subuni s. In a ecen s udy [1] he bio in binding ac i i y o hese p o eins
a e exposu e o ha sh condi ions like pH alues anging om 1 o 13 was measu ed by using
enzyme-linked bio in as a p obe. In con as o he p e ious s udy whe e he p o ein ac i i y was
measu ed a nea physiological pH condi ions–jus a e he ha sh en i onmen al ea men —we now
in es iga ed he abili y o a idin/s ep a idin/chime ic a idin o bind exac ly a ex eme pH alues.
This allows ge ing insigh s in o po en ial applica ions a ha sh condi ions.
Bio in is known o dissocia e om a idin and s ep a idin o e coming h ee o wo ene gy
ba ie s [9,10]. The highes ba ie he e ac s limi ing o he dissocia ion kine ics whe eas he lowe
ba ie s can be seen as semi-s able in e media e s a es, s abilized by newly o med weak bonds while
dissocia ion. Al hough nume ous echniques like su ace plasmon esonance o qua z c ys al
mic obalance a e a ailable o de e mine he a e aged unbinding beha io o bio in om immobilized
a idins, he explo a ion o he ene gy landscape equi es a single molecule echnique. Beside he
su ace o ce appa a us [11,12], op ical o magne ic weeze s [13], and bio-memb ane o ce p obe [14],
single molecule o ce spec oscopy (SMFS) is op imally sui ed o his pu pose. (S ep )a idin–bio in
was one o he i s ecep o –ligand sys ems in es iga ed wi h his echnique [15–18] and is s ill in he
ocus o ecen esea ch. In ea lie s udies changes on his ene gy landscape in oduced by p o ein
Molecules 2014, 19 12533
mu a ions [19–21] and by a ying en i onmen al condi ions [22,23] we e in es iga ed. Fu he mo e
e ec s o mul iple bio in binding [24,25], imp o emen s in measu emen [26] and in e p e a ion [27]
and simula ions o he sys em we e published [28–30]. SMFS allows measu ing he up u e o ces
unde di e en o ce loads, and, acco ding o E ans heo y, enables isualiza ion o possibly exis ing
di e en ene gy ba ie s yielding in he pa ame e s o he ene gy po en ial (i.e., ko and xβ). Wi hin his
s udy we applied AFM imaging and o ce spec oscopy o e eal possible changes o he ene gy
landscape o hese p o eins in he p esence o s ongly di e en H+ concen a ions anging om
pH 1 o 12.75.
2. Resul s and Discussion
2.1. Tip and Suppo Func ionaliza ion
Pe o ming single molecule o ce spec oscopy expe imen s equi e bo h s able e he ing o he
ligand o he ip as well as s able coupling o he co esponding ligand o he solid suppo . To ensu e
igh binding co alen chemis y is bes sui ed. Thus, we silanized mica using he APTES gas phase
p o ocol [31] esul ing in eac i e amino esidues. A sho homo bi- unc ional amino- eac i e linke
was used o liga e a idin p o eins o he su ace ia amino g oups. In Figu e 1 he amino-acids in he
β-ba el esponsible o bio in binding o (a) a idin, (b) chime ic a idin, and (c) s ep a idin a e
shown. In all cases he co alen coupling is pe o med by using lysine esidues on he ou e p o ein
su ace. To es he binding p o ocol, he p o ein unc ionalized su aces we e imaged using cons an
o ce con ac mode imaging wi h a so can ile e o a oid dena u a ion due o oo high inden a ion
o ces. A e imaging an a ea o 2.5 × 2.5 µm he scan size was educed o 0.5 × 0.5 µm and he
inden a ion o ce was inc eased signi ican ly o emo e he p o eins. As a esul , he heigh di e ences
be ween he p o ein su ace heigh le el and hose o he sc a ched a eas go isible. As shown in
Figu e 1 (lowe pa ) he heigh o (d) he a idin laye was 2.07 ± 0.47 nm, o (e) chime ic a idin
2.03 ± 0.15 nm, and ha o ( ) s ep a idin 1.86 ± 0.26 nm. The obse ed heigh s we e somewha
lowe compa ed o hose expec ed by he 3D s uc u es, which can be explained by he applied
inden a ion o ce o he AFM ip esul ing in a comp ession o he p o eins. Al hough mino
di e ences especially in he su ace oughness a e e iden i is p o en ha he coupling yielded in a
dense p o ein laye usable o u he SMFS measu emen s. Fo e he ing bio in o he ou e ip apex
a well-es ablished p o ocol as depic ed in Figu e 2a based on APTES gas phase silaniza ion o
silicon ni ide ips ollowed by co alen binding o he he e o bi- unc ional poly(e hyleneglycol)
c osslinke NHS-PEG(18)-bio in [32] was used.
2.2. Fo ce Dis ance Cycles, Da a E alua ion and Speci ici y P oo Expe imen s
To measu e he in e ac ion o ces be ween bio in and a idin o ce dis ance cycles (FDCs) we e
pe o med (Figu e 2b). The bio in- unc ionalized ip was app oached o he su ace ( ed line) wi hou
obse able bending o he can ile e . F om he momen o con ac , u he app oaching esul ed in an
upwa ds bending o he can ile e which was s opped a a p e iously se o ce limi o ypically
200–400 pN.
Molecules 2014, 19 12534
Figu e 1. (a) Molecula s uc u e o D-bio in in he β-ba el a idin (PDB 2AVI). (d) AFM
image o a co alen ly bound a idin laye . Sc a ching o a 0.5x0.5 µm a ea esul s in a hole.
In he c oss-sec ion below (co esponding o he yellow line in he AFM image) he heigh
di e ence o 2.07 ± 0.47 nm can be seen be ween a idin and he suppo . (b) Molecula
s uc u e o D-bio in in he β-ba el chime ic a idin p epa ed by posi ioning he ligand o
he apo s uc u e o chime ic a idin (PDB 3MMO) by using a idin-bio in complex as a
empla e. (e) AFM image o a co alen ly bound chime ic a idin laye . Image size is
2.5 × 2.5 µm, z-scale ba is 7 nm. The heigh di e ence in he c oss-sec ions is 1.86 ± 0.26 nm.
(c) Molecula s uc u e o D-bio in in he β-ba el s ep a idin (PDB 1MK5). ( ) AFM
image o a co alen ly bound s ep a idin laye . Image size is 2.5 × 2.5 µm, x-scale ba is
1 µm. The heigh di e ence in he c oss-sec ions is 2.03 ± 0.15 nm. The molecula
ep esen a ions we e p epa ed by using he p og am VMD.
In he e ac ion pe iod (black line) he can ile e bending ge s educed and, in he momen o
losing con ac , inds i s es ing posi ion again. In he whole pe iod o con ac bio in was able o o m a
complex wi h a idin, s ep a idin o chime ic a idin. In case o complex o ma ion a second – his
ime downwa ds – bending appea s as highligh ed by an a ow in Figu e 2b. The non-linea inc ease o
o ce while wi hd awing he can ile e wi h a cons an pulling eloci y is a esul o he s e ching o
he PEG e he , acco ding o he wo m-like-chain model. Mos impo an he up u e (unbinding) o ce
(i.e., he di e ence be ween he maximum downwa d bending and he es ing posi ion) can be
de e mined di ec ly by ansla ing he bending acco ding o Hook’s law in o a o ce. In case o no
Molecules 2014, 19 12535
complex o ma ion (as shown in Figu e 2b inse ) his ypical second bending e en does no appea .
The unbinding o ce di e s sligh ly a he same se ings as a esul o he he mal ene gy con ibu ion.
Thus, su icien s a is ics a e necessa y, which is ealized by epea ing he expe imen a leas 1,000 imes.
Figu e 2. (a) Tip and suppo chemis y. Uppe pa : APTES unc ionalized AFM ips a e
eac ed wi h he he e obi unc ional PEG e he NHS-PEG-bio in esul ing in a co alen
amide bond o ma ion. Lowe pa : a idin (o a idin-like p o ein) is co alen ly bound o
APTES coa ed mica using a homo-bi unc ional EGS c osslinke . (b) Typical o ce dis ance
cycle. The dis ance dependen can ile e bending is shown in ed o he app oaching
pe iod and in black o he e ac ion. In he la e a ypical unbinding e en , isible as
pa abolic shaped downwa ds bending can be seen (highligh ed wi h an a ow). In con as ,
he inse ep esen s a o ce dis ance cycle wi hou any speci ic in e ac ion. (c) Speci ici y
p oo exempla y shown o s ep a idin a a pulling eloci y o 400 nm/s a pH 7. The
black line ep esen s he p obabili y densi y unc ion using a bio in e he ed ip, whe eas
he ed line shows he p obabili y densi y unc ion o he e y same sys em a e blocking
he ip by adding ee s ep a idin.

Molecules 2014, 19 12536
By plo ing he p obabili y densi y unc ion (PDF) [33] which can be seen as e o weigh ed
his og am o he up u e o ces, he mos p obable unbinding e en can be de e mined. In Figu e 2c
black line a PDF cons uc ed om FDCs o s ep a idin and bio in measu emen s (a a pulling eloci y
o 400 nm/s and a pH 7) is shown wi h a mos p obable up u e o ce a 31.3 pN.
Al hough he shapes o he unbinding e en s in he FDCs gi e a clea hin ha he up u e is caused
by pulling on he PEG e he ed ligand om he su ace, a speci ici y p oo is needed o exclude
nonspeci ic binding o he ligand o he su ace. Fo his, we epea ed he expe imen wi h he e y
same ip and su ace bu pe o med a ip block by incuba ion o he ip in a solu ion o “ ee”
s ep a idin be o e he expe imen s. As a esul he binding p obabili y d opped down om 11.2%
be o e he block (Figu e 2c, black line) o 1.4% a e he block (Figu e 2c, ed line). PDFs a e se in
ela ion o hei binding p obabili y. The same p oo expe imen was pe o med o a idin (da a no
shown) esul ing in 15.5% be o e and 1.2% a e he block and o chime ic a idin (da a no shown)
yielding in a dec ease om 13% o 1.7%. Thus, i can be gua an eed ha all measu ed in e ac ion
o ces co espond o highly speci ic in e ac ions.
2.3. In luence o he pH Value on he Binding P obabili y
In o de o de e mine he in luence ega ding he pH on he binding p obabili y o bio in wi h
a idin/s ep a idin/chime ic a idin we pe o med SMFS expe imen s a di e en pH alues. To ha e
compa able esul s all da a se s we e acqui ed wi h he e y same ip and also by using he same
p o eins immobilized on a solid su ace. In all cases he measu emen s we e done beginning wi h
neu al pH and ended wi h he ha shes condi ions. To p o e ha he ac i i y o he a idin like p o eins
was no a ec ed by he p e ious condi ions (pH ea men ) we pe o med FDCs a ha sh pH condi ions
and a e wa ds exchanged he bu e back o neu al and again de e mined he binding beha io
owa ds bio in. Fo all p o eins i was shown ha e en a e exposu e (e.g., pH 12.75, whe e no s able
complex o ma ion could be obse ed o all p o eins) he ac i i y was eco e ed comple ely a e
changing back he pH alue o se en. The mean binding p obabili y a pH 7 was 17 ± 7.5% (n = 3)
which d opped a e changing he bu e a pH 12.75 o 0.4 ± 0.25% (n = 3), whe eas by changing back
o a pH 7 bu e he binding abili y eco e ed again nea ly o he same alue as be o e (13.8 ± 1.30%).
Thus we can conclude, ha no dena u a ion was obse able. To allow compa able esul s all
men ioned binding p obabili ies (i no s a ed o he wise) a e acqui ed a pulling eloci ies anging om
50–300 nm/s (i.e., 50, 100, 200, 300 nm/s). To ensu e ha he eco e ed p obabili y is no caused by
unspeci ic in e ac ion, we pe o med speci ici y p oo measu emen s by blocking he bio in on he ip.
As a esul he binding p obabili y d opped down o 1.4 ± 0.13% (n = 3) and hus success ully e i ied
he high speci ici y o he in e ac ion. As o de e mine he binding p obabili y o e a b oad pH ange
we pe o med single molecule o ce spec oscopy a pH alues anging om 1 o 12.75. Since we did
no expec a lowe ing in binding pe o mance a gen ly condi ions we only measu ed pH 7 as a
e e ence and ocused on pH alues o 3 and lowe as well as o 11 and highe (Figu e 3 and Table 1).
The abili y o a idin o bind bio in was e y compa able o e he ange om pH 2 o pH 11. A lowe
H+ concen a ions he p obabili y d opped down signi ican ly. In con as , he binding p obabili y o
bio in owa ds a idin signi ican ly inc eased a pH 1 (46.43 ± 7.58%, n = 4). I can be excluded, ha
his is only a esul o cha ge d i en inc eased adhesion since he speci ici y p oo by blocking he ip
Molecules 2014, 19 12537
e he ed bio in esul ed in a clea dec ease o he binding p obabili y (5.4%, n = 1) and hus e i ied
he speci ici y.
Figu e 3. (a) Compa ison o he binding p obabili ies o D-bio in wi h a idin (g ay),
chime ic a idin ( ed), and s ep a idin (blue) a di e en pH alues. (b) Complex li e imes
τ o a idin-bio in (g ay) and chime ic a idin-bio in ( ed) a di e en pH alues.
(c) Loading a e dependence o he mos p obable unbinding o ce o a idin (le ) and
chime ic a idin ( igh ) a pH alues anging om 1–11.
Table 1. Kine ic o - a es (ko ) and wid h o ene gy ba ie s (xβ) o a idin and chime ic
a idin a a loading a e egion anging om 100–10,000 pN/s.
A idin
ko [s-1] xβ [Å] Chime ic a idin
ko [s-1] xβ [Å]
pH 1 2.08 ± 0.08 4.18 ± 0.04 0.76 ± 0,05 7.31 ± 0.08
pH 2 3.00 ± 0.18 3.59 ± 0.07 2.10 ± 0.12 4.01 ± 0.06
pH 3 3.93 ± 0.19 4.70 ± 0.07 2.13 ± 0. 18 5.12 ± 0.10
pH 7 2.37 ± 0.14 4.49 ± 0.07 2.74 ± 0.18 4.29 ± 0.08
pH 11 0.88 ± 0.07 4.52 ± 0.08 2.22 ± 0.13 3.89 ± 0.06
One possible explana ion o his obse a ion could be a change in he con o ma ion o he loop
L3,4 in a idin. This loop ac s as a ga ekeepe in a idin, and is p obably hea ily esponsible o he
Molecules 2014, 19 12538
e y slow dissocia ion o bio in om a idin. Al hough apo-a idin s uc u es indica e lexible
con o ma ion o he loop [34,35], i is possible, ha L3,4 has signi ican nega i e impac on he
associa ion eloci y. The e o e, ou da a sugges ha a idin exhibi s a mo e open con o ma ion a low
pH, which, oge he wi h cha ge-media ed e ec s, acili a es apid bio in binding. Ve y high pH alues
esul ed in a comple e abolishmen o he binding (pH 12: 0.75 ± 0.84%, n = 4; pH 12.75:
0.43 ± 0.15%, n = 4). Chime ic a idin shows compa able esul s a pH 7 (12.68 ± 2.40%, n = 4) and 11
(19.83 ± 5.5%, n = 4), whe eas a low pH alues he p obabili y i s sligh ly dec eases a pH 3
(10.33 ± 5.39%, n = 4) bu inc eases again al eady a pH 2 (22.00 ± 7.83%, n = 4) and has a
signi ican ly highe alue a pH 1 (25.68 ± 4.84%, n = 4) compa ed o pH 7. In con as , he binding
p obabili y o s ep a idin owa ds bio in shows o he pH dependen beha io . A neu al pH
s ep a idin shows he highes binding p obabili y o 23.78 ± 6.98% (n = 4) owa ds bio in whe eas,
lowe ing and inc easing o he pH esul ed in a diminishmen o he p obabili y o complex o ma ion.
Bo h, he binding p obabili y a pH 11 (10.13 ± 6.88%, n = 4) as well as a pH 3 (9.60 ± 3.60%, n = 4)
was signi ican ly lowe ed. pH 2 (7.80 ± 7.53%, n = 4) and pH 1 (4.88 ± 1.22%, n = 4) esul ed in e en
lowe alues o he p obabili y o s ep a idin-bio in complex o ma ion bu s ill highe han pH 12
(3.13 ± 3.83%, n = 4) and pH 12.75 (0.38 ± 0.33%, n = 4).
Summed up, we could demons a e he abili y o a idin, s ep a idin and chime ic a idin o
speci ically bind bio in a ha sh condi ions o he i s ime a he single molecule le el. By looking
in o de ail, hey show clea di e ences in hei unc ionali y a he di e en en i onmen al condi ions
s udied. A idin and i s mu an chime ic a idin show a signi ican inc eased binding ac i i y a e y
low pH alues, whe eas he s uc u ally di e en s ep a idin has i s highes binding p obabili y a
pH 7, which ge s somewha linea ly lowe ed when changing he pH in bo h di ec ions. Thus, we
sugges he use o s ep a idin only a mode a e pH condi ions, whe eas a idin seems o be pe ec ly
sui ed o an ex emely acidic en i onmen . Chime ic a idin wo ks ine o e a b oad ange and
shows compa able binding beha io a pH 1 and pH 11. I has o be men ioned ha , in con as o
ea lie s udies, all gi en da a ep esen he ac i i y a (and no a e exposu e o ) hese ha sh
en i onmen al condi ions.
2.4. pH Induced Changes o he Bond Ene gy Landscape
A key ad an age o single molecule expe imen s is he possibili y o e eal he ene gy landscape o
an in e ac ion, especially i in e media e s a es appea in he unbinding p ocess. Bo h a idin and
s ep a idin a e known o ha e such s a es yielding in wo (s ep a idin) o h ee (a idin) ene gy
ba ie s, isible in he loading a e dependence o he unbinding o ce. Each change o slopes in his
plo co esponds o a di e en ba ie acco ding o E ans heo y [10] and Bells model [36]. No all
egimes o loading a es a e accessible by AFM, hence we show and discuss jus one ene gy ba ie
he e (i.e., be ween 100 and 10,000 pN/s) since o bo h, lowe and highe ene gy ba ie insu icien
ange o he loading a e is accessible o pe o m accu a e i ing. In dynamic o ce spec oscopy
expe imen s, we compa e a idin wi h i s mu an chime ic a idin. Fo bo h p o eins a pH7 , we epo a
ba ie a xβ ≈ 4Å (i.e., a dis ance o 4Å be ween he ee-ene gy minimum and he maximum o he
po en ial, measu ed in pulling di ec ion), which coincides wi h he in e media e s eng h egime o
a idin published by Me kel [10], De Pa is [18], and Taninaka [23]. This ba ie mos likely cons i u es
Molecules 2014, 19 12539
an in e media e ansi ion along he sepa a ion pa hway, as he he modynamically ele an ansi ion
s a e is gene ally assigned o he low o ce egime [37]. I should be men ioned ha ecen al e na i e
heo ies allow a di e en in e p e a ion o his beha io [27,38]. Thus, we also e alua ed he da a
ollowing he app oach o F iddle and Noy [27,38]. Fi ing he da a o a idin-bio in a pH 7 acco ding
o F iddle and Noy esul ed in a xβ o 4.27 Å, which is in good ag eemen , bu bo h ko
(5.33 s−1) and he equilib ium o ce eq (21.06 pN) di e ed signi ican ly om hei e alua ion o
Me kels’ [10] a idin-bio in da a (ko = 0.75 s−1, eq = 6.1). Mos p obably he limi ed ange o loading
a e o ou da a does no allow accu a e i ing o his model, hus we abandoned his i o ou da a.
The ko alues o he ba ie co esponding o he in es iga ed loading a e egion (102–104 pN/s) a y
signi ican ly in p e iously published wo k [10,18,19,25,39], anging om 0.08 s−1 [19] o 13.07 s−1 [25].
Ou esul s o ko o his a idin-bio in ene gy ba ie a pH 7 is 2.37 ± 0.14 s−1 and hus in be ween
he b oad ange o p e iously published esul s. I has o be men ioned ha he o - a e co esponding
o his ba ie does no e lec he mac oscopic dissocia ion kine ics, which a e expec ed o be
signi ican ly slowe . Ne e heless, ou esul s show he in luence o pH on his ba ie o he bond
ene gy landscape acco ding o E ans in e ms o kine ic o - a es (ko ) and alues o xβ shown in
Table 1. The co esponding loading a e dependence o he unbinding o ce is shown in Figu e 3c o
a idin (le ) and chime ic a idin ( igh ). The li e ime o a ansi ion s a e is gi en by he in e se
o - a e (τ = 1/ko ). Figu e 3(b) shows he ansi ion s a e li e imes o bo h in es iga ed p o eins a
di e en pH alues. Fo a idin a neu al pH, his ansi ion has a li e ime o τ = 0.42 s. In a mo e
acidic en i onmen , he li e ime is educed o τ = 0.25 s a pH 3, hen ises o τ = 0.33 s a pH 2 and
τ = 0.48 s a pH 1. Mos s iking is he inc eased ansi ion s a e li e ime a pH11 o τ = 1.14 s.
Chime ic a idin shows a simila li e ime a pH 7 (τ = 0.36 s), a ying he pH in bo h di ec ions has
also only mino e ec s, despi e a d as ic change a pH 1. In his ex eme en i onmen , he li e ime
ises o τ = 1.32 s.
3. Expe imen al Sec ion
3.1. Ma e ials
3.1.1. Chemicals
3-Aminop opyl ie hoxysilane (APTES; Sigma Ald ich, Vienna, Aus ia) was dis illed a low
p essu e and s o ed unde a gon in sealed c imp ials o e silica gel ( o a oid polyme isa ion) a
−20 °C. MilliQ pu i ied wa e (Millipo e, Bille ica, MA, USA) was used o all aqueous solu ions.
T ie hylamine (TEA, Sigma Ald ich) was s o ed unde a gon in he da k o a oid amine oxida ion. The
he e obi unc ional c osslinke Bio in-PEG-NHS was used as desc ibed p e iously [31]. Chlo o o m
was pu chased om J.T. Bake (G iesheim, Ge many), and a gon and N2 gas om Linde Gas GmbH
(S adl-Pau a, Aus ia). Musco i e mica shee s we e supplied by Ch is iane G öpl Elec on Mic oscopy
(Tulln, Aus ia). A idin and s ep a idin we e ob ained om Sigma-Ald ich. Gene a ion, pu i ica ion
and cha ac e iza ion o chime ic a idin we e published p e iously [1,2].
Molecules 2014, 19 12546
40. Hu e , J.L.; Bechhoe e , J. Calib a ion o a omic- o ce mic oscope ips. Re . Sci. Ins um. 1993,
64, 1868–1873.
41. Kam uzzahan, A.S.M.; Kienbe ge , F.; S oh, C.M.; Be g, J.; Huss, R.; Ebne , A.; Zhu, R.;
Rankl, C.; G ube , H.J.; Hin e do e , P. Imaging mo phological de ails and pa hological di e ences
o ed blood cells using apping-mode AFM. Biol. Chem. 2004, 385, 955–960.
42. Wildling, L.; Rankl, C.; Haselg üble , T.; G ube , H.J.; Holy, M.; Newman, A.H.; Zou, M.-F.;
Zhu, R.; F eissmu h, M.; Si e, H.H. P obing binding pocke o se o onin anspo e by single
molecula o ce spec oscopy on li ing cells. J. Biol. Chem. 2012, 287, 105–113.
Sample A ailabili y: The NHS-PEG-Bio in linke is comme cially a ailable, you can ge i also ia ou
ins i u e om P o . He mann G ube (h p://www.jku.a /biophysics/con en /e54633/e54706/e201881).
Also he A idin and S ep a idin a e comme cially a ailable and can be b ough om Sigma-Ald ich.
Chime ica idin is no pu chasable, we go his p o ein mu an om ou pa ne in Finland, Vesa
Hy önen and Ma kuu Kulomaa om he Uni e si y o Tampe e.
© 2014 by he au ho s; licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle
dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion license
(h p://c ea i ecommons.o g/licenses/by/3.0/).