AUTHOR QUERY FORM

Journal: BBAGEN Please e-mail or fax your responses and any corrections to:Chaiprasert-Paguio, JoslynE-mail: Corrections.ESSD@elsevier.spitech.comFax: +1 619 699 6721

Article Number: 27582

Dear Author,

Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-screen anno-tation in the PDF !le) or compile them in a separate list. Note: if you opt to annotate the !le with software other than AdobeReader then please also highlight the appropriate place in the PDF !le. To ensure fast publication of your paper please returnyour corrections within 48 hours.

For correction or revision of any artwork, please consult http://www.elsevier.com/artworkinstructions.

Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by "ags inthe proof. Click on the ‘Q’ link to go to the location in the proof.

Location in article Query / Remark: click on the Q link to goPlease insert your reply or correction at the corresponding line in the proof

Q1 Please con!rm that given names and surnames have been identi!ed correctly.

Q2 The given sentence seems to be incomplete. Please check for missing words/phrases and completethe sentence.

Q3 Please supply the year of publication.

Please check this box if you have nocorrections to make to the PDF !le. !

Thank you for your assistance.

Our reference: BBAGEN 27582 P-authorquery-v11

Page 1 of 1

UNCO

RRECTED P

RO

OF

1 Highlights

2 Biochimica et Biophysica Acta xxx (2013) xxx–xxx

4

5 Hofmeister ions control protein dynamics67 Balázs Szalontai a, Gergely Nagy b,c, Sashka Krumova d, Elfrieda Fodor e, Tibor Páli a, Stefka G. Taneva d, Gy!z! Garab f,8 Judith Peters c, András Dér a,!

910

a Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary11

b Wigner Research Centre for Physics, Institute for Solid State Physics and Optics, Hungarian Academy of Sciences, P.O. Box 49, H-1215 Budapest, Hungary12

c Institut Laue–Langevin, BP 156, 38042 Grenoble Cédex 9, France13

d Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl.21, 1113 So!a, Bulgaria14

e Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary15

f Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary

16

1718

19• Hofmeister anions affect the magnitude of protein conformational !uctuations.20• Accordance with a thermodynamic theory based on protein–water interfacial tension.21• Proof for the interplay between interfacial properties and conformational stability.

22

23

Biochimica et Biophysica Acta xxx (2013) xxx

BBAGEN-27582; No. of pages: 1; 4C:

0304-4165/$ – see front matter © 2013 Published by Elsevier B.V.http://dx.doi.org/10.1016/j.bbagen.2013.05.036

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbagen

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

Fig. S1 Original infrared absorption spectra of the three Hofmeister salt-treated bacteriorhodopsin samples in the Amide Iregion.The variations in absorbance represent the variations of the sample thickness between the CaF2 windows. The in-crease of the absorption at high temperatures is most probably due to the !(II)!!(I) transition, since its appearance coin-cides with the starting downshift of the Amide I band maximum. The latter is due to the decreasing contribution of the !(II)structure at 1667 cm!1, and the increasing !(I) contribution at 1652 cm!1.This would imply that the infrared absorptionextinction coef"cient is higher for !(I) than for !(II), but for this we do not have any proof, and such a problem is beyondthe scope of the present study. Nevertheless, this phenomenon does not distort the spectra and has no consequence on ourconclusions.

Fig. S2 A— Secondary structure-related changes in the Amide I region of bR upon increasing temperatures in the presence of0.5 M NaCl (Hofmeister neutral salt); B — in the presence of 0.5 M chaotropic NaClO4. The difference infrared absorptionspectra were obtained by subtracting the "rst spectrum recorded at the lowest temperature from the later ones. The lowerpanels show the temperature dependence of the minimum (1667 cm!1), and the two maxima (1652 and 1622 cm!1) ofthe spectra depicted in the corresponding upper panel. Note, that "rst !(I) starts to grow at the expense of !(II), then both!-structures diminish into the emerging "-band, which is indicative of irreversible protein denaturing.

Fig. S3 Original infrared absorption spectra of the three Hofmeister salt-treated bacteriorhodopsin samples in the Amide IIregion.Note that while the absorbances of the bR-NaCl and bR-NaClO4 samples are very close, and each has its maximumat around 1546 cm!1, the bR-NaCOOCH3 sample has about ten times higher absorption and its maximum is at around1560 cm!1. This is due to the #COO! vibration of the acetate ions. The break-down of the Amide II bands at high temper-atures is due to the H/D exchange, the deuterated peptide groups have their Amide II" vibrations at around 1450 cm!1,therefore the Amide II band of the protonated peptide groups disappears. This effect cannot be seen for the bR-NaCOOCH3 sample because of the overlap of the intensive #COO! band. However, as it is shown below in Fig. S3, singularvalue decomposition (SVD) analysis can reveal the variations of the protein related Amide II band even in the presence ofthis huge, overlapping absorption here.

Fig. S4 Singular value decomposition (SVD) analysis of the three Hofmeister salt-treated bacteriorhodopsin samples. Note, asit could be seen already in Fig. S2, that both the average s1, and the change-related s2 spectra of the bR-NaCl and the bR-NaClO4 samples are similar.Q2 Surprisingly, while its s1 spectrum is different from, the s2 spectrum of the bRNaCOOCH3 is sim-ilar to that of the other two samples. This shows the power of the SVD analysis to reveal the changing components of a sys-tem. Using similar approach, we could earlier demonstrate the phase transition of a single lipid bilayer embedded into acomparatively large polyelectrolyte "lm architecture (Pilbat et al. (2007) Langmuir, 23:8236-8242).The temperature depen-dences of these s2 vectors are given in Fig. 5 of the manuscript.

UNCO

RRECTED P

RO

OF

1 Hofmeister ions control protein dynamics

2 BalázsQ1 Szalontai a, Gergely Nagy b,c,1, Sashka Krumova d, Elfrieda Fodor e, Tibor Páli a, Stefka G. Taneva d,2,3 Gy!z! Garab f, Judith Peters c,3, András Dér a,!4 a Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary5 b Wigner Research Centre for Physics, Institute for Solid State Physics and Optics, Hungarian Academy of Sciences, P.O. Box 49, H-1215 Budapest, Hungary6 c Institut Laue–Langevin, BP 156, 38042 Grenoble Cédex 9, France7 d Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl.21, 1113 So!a, Bulgaria8 e Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary9 f Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary

10

11

a b s t r a c ta r t i c l e i n f o

12 Article history:13 Received 16 April 201314 Received in revised form 24 May 201315 Accepted 28 May 201316 Available online xxxx17181920 Keywords:21 Hofmeister effect22 Differential scanning calorimetry23 Neutron scattering24 Fourier transform infrared spectroscopy25 Protein structural !uctuation26 Bacteriorhodopsin

27Background: Recently, we have elaborated a thermodynamic theory that could coherently interpret the28diverse effects of Hofmeister ions on proteins, based on a single physical parameter, the protein–water interfacial29tension (Dér et al., Journal of Physical Chemistry B. 2007, 111, 5344–5350). This theory, implying a “liquid drop30model”, predicts changes in protein conformational !uctuations upon addition of Hofmeister salts (containing31either kosmotropic or chaotropic anions) to the medium.32Methods: Here, we report experimental tests of this prediction using a complex approach by applying methods33especially suited for the detection of protein !uctuation changes (neutron scattering, micro-calorimetry, and34Fourier-transform infrared spectroscopy).35Results: It is demonstrated that Hofmeister salts, via setting the hydrophobic/hydrophilic properties of the pro-36tein–water interface, control conformational !uctuations even in the interior of the typical membrane transport37protein bacteriorhodopsin, around its temperature-induced, unusual !(II) ! !(I) conformational transition38between 60 and 90 °C. We found that below this transition kosmotropic (COOCH3

!), while above it chaotropic39(ClO4

!) anions increase structural !uctuations of bR. It was also shown that, in each case, an onset of enhanced40equilibrium !uctuations presages this phase transition in the course of the thermotropic response of bR.41Conclusions: These results are in full agreement with the theory, and demonstrate that predictions based on42protein–water interfacial tension changes can describeHofmeister effects and interpret protein dynamics phenom-43ena even in unusual cases.44General signi!cance: This approach is expected to provide a useful guide to understand the principles governing the45interplay between protein interfacial properties and conformational dynamics, in general.46© 2013 Published by Elsevier B.V.

4748

49

50

51 1. Introduction

52 Hofmeister effects (HEs) cover a wide range of salt-induced phe-53 nomena on proteins, and on colloidal particles in general, including54 changes of solubility, denaturation, or enzyme kinetics. In his original

55work, Hofmeister [1] reported that certain salts (primarily their56anions) either decrease or increase protein solubility when present57in the solution at moderate or high concentrations (usually above58100 mM), and arranged them into the so-called Hofmeister series (HS)59according to the sign and the magnitude of the effect they exerted. The60HS of themost important anions, in descending order of their precipitat-61ing ability, is as follows: F! " SO4

2! > HPO42! > CH3COO! > Cl! >

62NO3! > Br! > ClO3

! > I! > ClO4! > SCN!. Members in the series left

63of the “Hofmeister-neutral” Cl! are precipitants, while the ones right64of it are solubilizers. An impressive number of follow-up studies show65that the same HS emerges in other phenomena, like denaturation of66proteins, inhibition or activation of enzymes, as well (for reviews, see67[2–4]). HEs live their renaissance in the past few years. Besides their68signi"cance in colloid chemistry, preparative biochemistry and biotech-69nology, they have been successfully applied in studying the function70and dynamics of macromolecular structures [5,6]. In addition, a recent71review calls the attention to their role in basic pathophysiologic issues72[7].

Biochimica et Biophysica Acta xxx (2013) xxx–xxx

! Corresponding author at: Institute of Biophysics, Biological ResearchCentre, HungarianAcademy of Sciences, H-6726 Szeged, Temesvári krt. 62., Hungary. Tel.: +36 62 599 606;fax: +36 62 433 133.

E-mail addresses: szalontai.balazs@brc.mta.hu (B. Szalontai), nagy@ill.fr,jpeters@ill.fr (G. Nagy), sakrumo@gmail.com (S. Krumova), fffrida7@gmail.com(E. Fodor), stefka.germanova@ehu.es (S.G. Taneva), der.andras@brc.mta.hu (A. Dér).

1 Present address: Paul Scherrer Institute, Laboratory for Neutron Scattering, 5232Villigen PSI, Switzerland.

2 Present address: Unidad de Biofísica (CSIC-UPV/EHU) and Departamento deBioquímica y Biología Molecular, Universidad del País Vasco, 48080 Bilbao, Spain.

3 Present address: University Joseph Fourier, UFR PhITEM, BP 53, 38041 GrenobleCédex 9, France; Institut de Biologie Structurale, 41 rue Jules Horowitz, 38027 GrenobleCédex 1, France.

BBAGEN-27582; No. of pages: 9; 4C: 4, 5, 6, 8

0304-4165/$ – see front matter © 2013 Published by Elsevier B.V.http://dx.doi.org/10.1016/j.bbagen.2013.05.036

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbagen

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

UNCO

RRECTED P

RO

OF

73 Despite the widespread occurrence of HEs, and the extensive74 research efforts focused on them, their interpretation has remained75 a matter of debate [8], because of the lack of a unifying formalism76 covering the entire spectrum of salts from “salting out” to “salting77 in” effects, i.e. from precipitants to solubilizers. One approach [9] cor-78 related these attributes with effects on water structure, in particular79 on the fraction of hydrogen-bonded water molecules: precipitants80 promote H-bond formation between the water molecules in their81 vicinity, and are therefore called kosmotropes, while solubilizers82 break H-bonds between water molecules, and are therefore called83 chaotropes. In another branch of interpretations, dispersion forces84 were suspected to be the main factor responsible for HEs [10].85 Although these are certainly among the main microphysical factors86 responsible for the Hofmeister effects, all attempts aiming to assign87 a single physical parameter as an approximate measure for the88 whole diversity of HEs, however, have failed for more than a century.89 In a recent work, we have demonstrated that a uni"ed, phenome-90 nological formalism is able to qualitatively account for the entire91 range of HE-related phenomena [11]. The crucial difference between92 our approach and the previous attempts of similar intention was93 that, instead of building on the conventionally used air–water inter-94 face [9], we took protein–water interfacial tension ($pw) as the prin-95 cipal physical parameter to describe Hofmeister effects. The most96 important conclusion of our theory is that HEs are manifested via97 the surface-dependent term of the free energy (Gs = $pw · ASA,98 where ASA is the solvent accessible surface area) of the proteins99 (or, in general, of colloid disperse systems) [12]. In other words,100 they modify the hydrophobic effect at protein–water interfaces [13].101 The addition of kosmotropic or chaotropic salts to the solvent102 increases or decreases the solute–water interfacial tension, respec-103 tively, and thus, Gs should increase or decrease accordingly. The104 alteration of Gs is the driving force behind the observed HEs105 affecting either the aggregation or the conformation of the proteins.106 Note that $pw should, necessarily, depend also on the quality of the107 solvent-exposed protein surface, and may even take negative values,108 too (e.g., in case of proteins of naturally open conformation) [11,14].109 An implication of our theory is that HEs are manifested by chang-110 ing transition rates and equilibrium constants in reactions accompa-111 nying major conformational changes that involve changes in the112 water-exposed surface area of macromolecules and supramolecular113 assemblies. (Such effects have recently been exempli"ed by a de-114 tailed study of Hofmeister effects on the function of photoactive115 yellow protein [5].) It was also established that interfacial tension and116 protein stability are interconnected by protein conformational !uctua-117 tions, thus providing the keystone for the microscopic interpretation118 of HEs [11,15]. In consequence of the above view, the same HS should119 appear for salt-induced protein conformational !uctuations as for pre-120 cipitation or conformational changes.121 In spite of the success of this theory in describing the diverse122 Hofmeister phenomena, so far direct experimental evidences for123 salt-induced changes of protein conformational !uctuations have124 been missing. Hence, here we used a complex experimental approach125 to monitor temperature-induced changes of protein structure and126 dynamics, with methods specially adapted for observing changes in127 conformational !uctuations (neutron scattering, differential scanning128 calorimetry (DSC), and Fourier transform infrared spectroscopy129 (FTIR)). Our model object was the prototypical retinal protein, bacte-130 riorhodopsin (bR) from Halobacterium salinarum [16]. Also known131 as the simplest ion pump in biological systems, bR is one of the132 best-characterized and most robust membrane proteins, subjected133 to non-speci"c anion binding effects. Despite, both its structure and134 function have been shown to be in!uenced by interactions with135 Hofmeister anions [11,17]. In the present study, special attention136 was paid to the reversible conformational change of bR in the course137 of its heat denaturation, the !(II) ! !(I) conformational transition138 between 60 and 90 °C, where enhanced changes of structural

139!uctuations were expected to arise [18,19], due to the associated140alterations of the solvent-exposed surface.141So far, there have been only a few reports about proteins having142an “open” conformation as their naturally most stable form [3], and143bR is considered to be such one. Namely, previous FTIR measurements144on bR detected !(II) helices below ca. 50 °C [18], and between 60 and14590 °C, bR undergoes an !(II) ! !(I) conformational transition [19].146In !(I), however, the helix is known to be more tightly packed than147in !(II) [18,20]. It implies that, in contrast to most proteins, here148the accessible surface area (ASA) of the protein is higher at lower149temperatures (!(II)), and ASA decreases upon the temperature-150induced transition of the secondary structure (to !(I)). For such a151case, our theory [11] predicts an unusual phenomenon to occur:152instead of kosmotropic, here chaotropic salts should have a stabilizing153effect; consequently, they are expected to shift the !(II) ! !(I) tran-154sition to higher temperatures, while kosmotropes are expected to155destabilize the open !(II) protein conformation, therefore shifting156the transition toward lower temperatures, as it has been indicated157by our former DSC experiments [11].We took advantage of this unusual158feature of bR to test the predictive power of our HE-interpreting theory.

1592. Materials and methods

1602.1. Bacteriorhodopsin-containing purple membrane (bR) isolation

161Purple membranes were isolated according to the standard proce-162dure [21]. Purple membranes were always re-suspended in D2O-based163buffers, due to the needs of neutron scattering and FTIR experiments164(10 mM HEPES in D2O at pD 6.6, containing 500 mM kosmotropic,165Hofmeister-neutral, or chaotropic salts, NaCOOCH3, NaCl, or NaClO4,166respectively, as required). The bR samples, treated with these salts167will be denoted as bR-NaCOOCH3, bR-NaCl, and bR-NaClO4 throughout168the paper.

1692.2. Neutron scattering experiments

170For neutron scattering experiments the suspensions of bR-containing171purple membranes were centrifuged for 20 min at 40,000 g. The pellets172were placed in !at rectangular aluminum sample holder with an area173(30 ! 40 mm2) adapted to the dimensions of the incident neutron174beam. The sample holders were hermetically closed and used for the175experiment.176The experiments were carried out on the IN13 backscattering177spectrometer at the Institut Laue Langevin (ILL, France). Two samples178were measured by elastic incoherent neutron scattering to determine179the atomic mean-square displacements bu2> as a function of temper-180ature. One of the samples was bR-NaClO4, measured in the 40–91 °C181temperature range, the other one was bR-NaCOOCH3 measured be-182tween 40 and 87 °C. Temperature scans were done upon increasing183the temperature in steps of 7–10 °C below the transition region, and184of 3–4 °C around the transition region. Additional vanadium, buffer185and empty cell measurements were recorded for correction and nor-186malization purposes [22].187In the examined time range (8 %eV energy resolution, correspond-188ing to a time window of 100 ps through Heisenberg's uncertainty189principle) on backscattering spectrometers, H-atom motions re!ect190the motions of the chemical groups to which they are bound [23].191The scattered elastic incoherent intensity can be described within the192Gaussian approximation [24] by

Iel Q ;! ! 0" "E# $#I0 exp !16

u2D E

Q2! "

#1$

193194where I0 is the intensity hitting the sample, bu2> is the average atomic195mean square displacement,# corresponds to the energy transfer, and Q196to the momentum transfer between neutron and target. The average

2 B. Szalontai et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

UNCO

RRECTED P

RO

OF

197 value of the atomic mean square displacements (MSD) for the protein198 can thus be obtained for each temperature from the slope of the199 semi-logarithmic plot of the incoherent scattering function through

u2D E

! !6d lnIel Q ;! ! 0" "E# $

dQ2 : #2$

200201202 The data treatment, including subtraction of the scattering from203 the empty sample holder, normalization to vanadium and absorption204 correction based on the correction formula of Paalman–Pings coef"-205 cients [25] was performed using the ILL program LAMP (Large Array206 Manipulation Program [26]).

207 2.3. Differential scanning calorimetry experiments

208 Differential scanning calorimetry (DSC) is a useful tool to charac-209 terize the thermodynamics of protein conformational changes [27],210 and has been applied to monitor the steps of thermal denaturation211 of bR, too [11,28,29]. Direct information about the speci"c heat of212 the protein (Cp) can be derived from the experiments, and Cp is213 instantaneously related to enthalpy !uctuations, according to the214 !uctuation-dissipation theorem [27]:

Cp ! b $H2 >

kT2 : #3$

215216217 DSCmeasurementswere performed on bRmembrane suspensions of218 6 mg·mL!1 protein concentrations, by using a VP-DSC high-sensitivity219 differential scanning calorimeter (MicroCal Inc., Northampton, MA)220 equipped with built-in twin reference and sample cells of 0.5 mL221 volume. Temperature scans were performed in the 10–110 °C range, at222 a heating rate of 0.5 °C·min!1, and the differential heat capacities223 were recorded. At the end of the heating scans samples were cooled224 back to 10 °C where they were allowed to equilibrate for 30 min before225 being subjected to a second heating scan with the same measuring pro-226 tocol. These re-heating scans served later as baselines for the "rst record-227 ings since they did not show any transition peak present in the above228 mentioned temperature range. Positions of the pre- and main transition229 peaks were determined by using the built-in integration routine of the230 DSC analysis software (MicroCal-Origin, DSC application).

231 2.4. Infrared measurements

232 FTIR spectra were recorded on a Philips PU9800 Fourier transform233 infrared spectrometer, averaging 128 scans at 2 cm!1 spectral resolu-234 tions. For the infrared experiments, 1.5 mL aliquots of bR-containing235 purple membranes dispersed in the required D2O-based buffer +236 Hofmeister salt, were concentrated in a Hettich (Germany) EBA 12R237 tabletop centrifuge (4 °C, 24,000 !g, 8 min). The pellet (about 20 %L)238 was placed between CaF2 windows separated by an aluminum spacer239 (15 %m). Temperature dependence of the infrared absorption spectra240 was measured by recording 35 ! 2 FTIR spectra between 5 and 95 °C241 in about 3 °C steps, leaving 7 min for reaching thermal equilibrium. At242 each temperature, two absorption spectra weremeasured by recording243 for each single beam background (empty spectrometer) and sample244 spectrum (CaF2 window plus purple membrane dispersion) by using245 a sample shuttle. A water-thermostated cell holder controlled the tem-246 perature of the sample. The accuracy of the temperature setting was247 about 0.1 °C.248 In water suspensions of most biological membranes, the Amide I249 band is centered at around 1650 cm!1, arising mainly from the250 C_O stretching vibration (with minor contributions from the251 out-of-phase C\N stretching vibration, the C\C\N deformation252 and the N\H in-plane bend). The Amide II mode near 1550 cm!1 is253 the out-of-phase combination of the NH in-plane bend and the CN

254stretching vibration with smaller contributions from CC, NC stretching255vibrations, and CO in-plane bend [30].256To keep the Amide I region free from the disturbing strong contri-257bution of the H2O-bending band at 1643 cm!1, and being in agree-258ment with the neutron scattering and DSC experiments, all infrared259spectra were recorded in D2O environment. The presence of D2O260induces the exchange of the peptide \NH groups to \ND ones,261which affects the Amide I and II regions differently. The H/D isotope262effect is weak in the Amide I region, since N\H vibrations have little263contribution to these modes, as discussed above (the shape of the264Amide I region does not change considerably, only the frequencies265of the component bands shift down by a few wavenumbers; the266deuterated Amide I band will be indicated throughout the paper as267Amide I"). Temperature-dependent changes in the Amide I" remain268characteristic to the changes of the secondary structure elements of269the protein.270To monitor protein dynamics, we used the Amide II region, where271H/D exchange exerts a major effect, since the Amide II region down-272shifts to about 1450 cm!1 (Amide II"). Therefore, the Amide II band273(around 1550 cm!1) disappears upon H/D exchange. The rate of274this disappearance is informative about protein structural !uctuations275[31,32], since the access of the external D atoms to the peptide \NH276groups depends on the protein dynamics under the given environ-277mental factors (temperature, molecular interactions).

2782.4.1. Singular value decomposition (SVD) analysis279Temperature-dependent changes in the infrared spectra were280studied by SVD analysis [33]. Brie!y, the D (n $ m) data matrix was281decomposed as D = S $ W $ VT, where n is the number of wave-282lengths, and m is the number of temperatures at which the measure-283ments were made. S (n $ m) contains the orthonormal abstract284spectrum vectors; the W (m $ m) diagonal matrix consists of the285singular values in descending order, while V (m $ m) contains the286orthonormal vectors describing the temperature dependence.287The s1 base spectrum (the "rst column of the S matrix) shows the288average spectrum over the temperature range involved; s2 shows the289largest changes accompanying the temperature increase, which have290to be combined with the s1 spectrum to get back the actual spectrum291at a given temperature. The v1 and v2 amplitude vectors give the292temperature dependence of the average, and the largest change,293respectively, manifested in the s1, s2 spectra, etc. In the present SVD294analysis, we considered only the average (s1, v1) and the largest295change (s2, v2) of the spectra recorded upon increasing temperature296(5–95 °C). The higher order SVD components (s3, v3, s4, v4…, etc.)297were neglected because their weights were low. This approach is298evidently an approximation, which, however, can be justi"ed since299the weights (wi) of the si base spectra were decreasing very rapidly300(to about 2–3% of w1 by w3, data not shown).301All calculations were carried out with the SPSERV© software of Cs.302Bagyinka (Biological Research Centre, Szeged, Hungary). For more303details on the use of SVD analysis on biological membrane proteins,304see our earlier work on dynamics and temperature-induced denatur-305ation of proteins in thylakoid membranes [34].

3063. Results

3073.1. Neutron scattering experiments

308Among the three applied methods, neutron scattering gives the309most direct information about conformational !uctuations. It probes310mainly the mean square displacements (MSD) of hydrogen atoms in311biological samples, since the incoherent cross-section of H is much312larger than that of any other atom in the samples. Since H atoms are313usually homogeneously distributed in biological samples, the MSD314are assumed to re!ect the global dynamics of the system as well.

3B. Szalontai et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

UNCO

RRECTED P

RO

OF

315 The bR-containing purple membranes consist of two types of316 molecules, bR and the lipid components of the membranes, therefore,317 in principle, we should see in the scattering experiments contribu-318 tions from both lipids and bR. Nevertheless, by choosing the temper-319 ature range where bR, but not the lipid, is expected to have a major320 structural transition, and taking into account the 75:25 weight ratio321 of bR to lipids in purple membranes, one can assume that the changes322 seen in H-dynamics are mostly due to H-atoms of bR, and that323 lipid-related H atoms provide, at most, a smoothly changing back-324 ground. A further factor to be taken into account is the H/D exchange325 at the \NH groups of the solvent accessible peptide bonds. This326 means that especially at higher temperatures, the decreasing popula-327 tion of non-D2O-accessible peptide groups, which are either in the328 interior or at the lipid interface of bR, will have the major contribution329 to the neutron scattering signals, i.e., this method monitors the least330 accessible, internal parts of the protein.331 Bacteriorhodopsin dynamics has been studied by incoherent neu-332 tron scattering over the past decades [35–39]. In these experiments,333 two populations of motions have been found to arise when investigat-334 ing the thermotropic behavior of fully hydrated bR with neutron scat-335 tering. They were assigned to dynamical transitions around !123 °C336 and !23 °C, where onsets of additional conformational motions occur337 [37]. In the present case, however, wemeasured atmuch higher temper-338 atures (in the 40–91 °C range), in order tomonitor Hofmeister effects on339 protein !uctuations associated with the !(II) ! !(I) transition of bR.340 Neutron scattering experiments were carried out on bR-containing341 purple membranes suspended in D2O-based kosmotropic acetate and342 chaotropic perchlorate salt solutions (500 mM NaCOOCH3 and NaClO4,343 respectively).344 In Fig. 1, MSD values of hydrogen atoms are plotted as a function345 of temperature. They are associated with the sample !exibility via the346 !uctuation-dissipation theorem [40]. At higher temperatures, where347 the solvent of the protein is !uid, the elastically scattered intensity348 decreases drastically, as the motions are leaving the instrumental time349 window. This fact leads to high errors (compare our errors bars with350 those in Ref. [41]) of the calculated MSD values. Nevertheless, a break in

351the slope of the MSD(T) function for kosmotrope-treated bR-NaCOOCH3

352around 75 °C, and for the chaotrope-treated bR-NaClO4 around 80 °C is353clearly visible. The mean force constants, which are associated with the354stability of the whole sample, can be separately calculated for the two

355temperature regions according to the kh i ! 0:00276d u2h i=dT formula [36,42]. The

356"tted straight lines and their slopes of the obtained MSD values and the357force constants derived from their slopes are shown in Fig. 1. Note, that358the chaotropic sample below the transition has a higher mean force con-359stant (a lower slope of the "tted curve) associatedwith a lower !exibility360as compared to that of the kosmotropic sample. Above the transition,361however, the calculated !k" is slightly lower for the chaotropic sample362than for the kosmotropic one, but the difference is within the error of363the measurements. In other words, below the transition the resistance364against structural !uctuations is higher (i.e., the conformational !exibility365is lower) in bR-NaClO4 than in bR-NaCOOCH3, while above the transition,366they are about the same in the two cases (or might even be oppositely367related). This implies a higher “dynamical stability” of bR-NaClO4 than368that of bR-NaCOOCH3, being also re!ected by the up-shift of the transition369temperature of bR by the chaotropic salt.

3703.2. Calorimetric measurements

371In order to correlate !uctuation changes with thermodynamic372transitions, complementary differential scanning calorimetry (DSC)373experiments were performed under similar ambient conditions (i.e., in374D2O-based media). For the DSC measurements, however, the set of salt375solutions was extended by the Hofmeister-neutral chloride, in order to376allow a full comparison with results obtained earlier with H2O-based377salts [11], too.378Fig. 2 shows that the main unfolding transition peak of the Cp(T)379curve is located at around 100 °C, slightly depending on the particular380Hofmeister salt, while a minor peak (the so-called “pre-transition”)

Fig. 1. MSDs as the function of temperature for bR-containing purple membranessuspended in D2O-based 0.5 M kosmotropic (NaCOOCH3) and chaotropic (NaClO4)Hofmeister salt solutions. Straight lines represent linear "ts used to extract the forceconstants bk>. A change in the slope of the MSD values can be observed at around75 °C for bR-NaCOOCH3, and at around 80 °C for bR-NaClO4. The lower conformational!exibility below the transition and the higher transition temperature in bR-NaClO4

compared to bR-NaCOOCH3 suggest a higher dynamical stability for the chaotrope-treatedsample as compared to the kosmotrope-treated one.

Fig. 2. DSC heating endotherms of bR-containing purple membranes suspended inD2O-based solutions of 0.5 M of various Hofmeister salts. Temperature pro"les ofexcess molar heat capacities from top to bottom correspond to samples in the presenceof chaotropic NaClO4 (red), neutral NaCl (green), and kosmotropic NaCOOCH3 (blue),respectively. The two vertical lines indicate the temperature range, in which the!(II) ! !(I) transition of bR takes place in the various Hofmeister salts. The numbersindicate the critical temperatures (Tc) associated to the reversible !(II) ! !(I) transi-tion (“pre-transition”) and the irreversible (denaturing) maxima. The scanning ratewas 0.5 °C·min!1. The data were subjected to baseline subtraction by using the secondheating scans as baselines, and were normalized to the corresponding protein concen-trations. For better visibility, theNaClO4 andNaCOOCH3 curveswere up- anddownshifted,respectively.

4 B. Szalontai et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

UNCO

RRECTED P

RO

OF

381 appears in the 75-90 °C range. In all cases, transitions could not be382 detected in the second heating scans, indicating that during the "rst383 scan a complete and irreversible heat-denaturation of the protein384 occurred. This indicates that the main transition peak of the Cp(T)385 curve most probably re!ects the "nal, irreversible denaturation step386 of the bR accompanied also by a decomposition of the purple mem-387 brane. Therefore, in the following we do not consider the analysis of388 this peak. From our point of view, much more interesting is the389 minor, reversible pre-transition in the 75–90 °C range, which exhibits390 strong Hofmeister salt dependence. This transition has been assigned391 to the !(II) ! !(I) conformational change [19,28]. The chaotropic392 ClO4

! ions shifted the critical temperature (Tc) of this transition up393 to ca. 88 °C, as compared to the Hofmeister-neutral Cl! around394 82 °C, while the kosmotropic CH3COO! ions downshifted the same395 transition to about 77 °C. It is worth mentioning that these values are396 by a few degrees higher than the ones measured on non-deuterated397 bR [11], and that the peaks related to the !(II) ! !(I) transitions are398 getting sharper when moving from the kosmotropic acetate to the399 chaotropic perchlorate (Fig. 2).400 From theCp(T) curves, themolar enthalpy andheat capacity changes401 (&H and &Cp values, respectively) associated with the !(II) ! !(I)402 transition were also determined according to Jelesarov and Bosshard403 [43] for bR suspended in the three different salt solutions (Table 1).404 The molar enthalpy changes were determined from the integral of405 the Cp(T) functions, and found to be the same for the three different406 salts within the error, implying that the main differences between407 the initial and product secondary structures of the protein (!(II) and408 !(I) states, respectively) are presumably the same in all the three409 cases. The corresponding &Cp values, determined from lines "tted to410 the Cp(T) functions before and after the transition, are close to zero411 within the temperature range of interest.

412 3.3. Fourier transform infrared (FTIR) experiments

413 To reveal the sequence and the ion dependence of the secondary414 structure changes in bR, FTIR experiments were carried out on different415 Hofmeister salt-treated bR samples. The salt content of the FTIR samples416 was the same as those of used in the neutron scattering and DSC mea-417 surements. The Amide I" and Amide II regions of the infrared spectra,418 characteristic of the secondary structure and dynamics of proteins,419 were recorded and analyzed.420 The sequence of the secondary structure changes (from !(II)421 through !(I) to "), could be examined by the temperature depen-422 dence of the evolution of the related infrared absorption bands in423 the difference spectra, obtained by subtracting the absorption spec-424 trum recorded at the lowest temperature from the later ones. Since425 all the three samples behaved qualitatively similar (see Fig. S1 in426 Supplementary data) we present, as an example, the evolution of427 changes in the Amide I" region upon temperature increase only for428 the kosmotrope-treated bR-NaCOOCH3 (Fig. 3). (The same results429 for the other two salts are given in Fig. S2.) In the difference spectra430 depicted here (Fig. 3A), "rst, a shallow valley around 1667 cm!1 is431 the only feature, which slowly deepens toward higher temperatures.432 In agreement with the literature, this frequency was assigned to433 !(II) helices [19,28]. Around 80–85 °C, the changes become more434 characteristic, partly due to the deepening of the 1667 cm!1 band,

435and partly due to an emerging band at around 1652 cm!1, assigned436to !(I) helices [44]. Above 90 °C, the strong, "-structure-related437band at around 1622 cm!1 dominates the difference spectra (one438may observe a weaker pair-band around 1685 cm!1 as well in Fig. 3A439as it should be [44,45]). This pair of bands was assigned earlier to440anti-parallel "-sheets [44]. The appearance of this type of "-structure441always accompanies the heat-induced denaturation of proteins [45].442For better visualizing the sequence of events, we have plotted just the443maxima of the three bands as a function of temperature in Fig. 3B.444From Fig. 3 onemay conclude that on theway to heat-induced denatur-445ation of bR, "rst the !(II) structure is transformed to !(I), then !(I)446(and the remaining !(II)) to "-sheets. The same sequence of events447could be established for bR-NaCl, and bR-NaClO4, as well, except that448the corresponding spectral changes started at higher temperatures449(shown in Fig. S2).

Table 1t1:1

t1:2 Molar enthalpy and heat capacity changes during the temperature-induced !(II) ! !(I)t1:3 transition of bacteriorhodopsin, and the corresponding critical absolute temperatures.

t1:4 bR-NaOOCH3 bR-NaCl bR-NaClO4

t1:5 &H (kcal·mol!1) 11.4 ± 0.5 9.7 ± 3.1 10.9 ± 0.4t1:6 &Cp(kcal·mol!1·K!1) 0.4 ± 0.5 !0.5 ± 0.5 !0.7 ± 0.5t1:7 Tc (°C) 77 ± 0.5 82 ± 0.5 88 ± 0.5

Fig. 3. Secondary structure-related changes in the Amide I" region of bR in the presenceof 0.5 M NaCOOCH3 (kosmotropic) Hofmeister salt upon increasing temperatures.A — Difference absorption spectra obtained by subtracting the "rst infrared absorptionrecorded at the lowest temperature from the later ones. B — The temperature depen-dence of the maxima of the three bands extracted from the spectra depicted in panelA. Note that "rst !(I) starts to grow at the expense of !(II), then both !-structuresdiminish into the emerging "-band, which is indicative of irreversible proteindenaturation.

5B. Szalontai et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

UNCO

RRECTED P

RO

OF

450 In order to give a more rigorous analysis of the similarities in the451 secondary structures of bR in the presence of different Hofmeister452 salts, and of the differences between the dynamics and stability of453 these structures upon heat-induced changes, we analyzed the tem-454 perature series of the recorded infrared spectra with SVD (Fig. 4).455 (The advantage of this pure mathematical procedure is that it does456 not require any model for the decomposition of the measured spectra,457 and uses the whole data set on the full spectral and temperature458 range investigated [33].) The s1 and s2 base spectra provide informa-459 tion about the spectral changes of the samples in the investigated460 temperature range.461 Fig. 4A shows the s1 base spectra (corresponding to the average462 structure of the temperature series) derived from the Amide I"463 regions of bR in the presence of different Hofmeister salts recorded464 between 5 and 93 °C. It can be seen that s1 spectra are very similar,465 i.e. the majority of the secondary structure elements contributing to

466the Amide I" region are not affected by the Hofmeister salts (in other467words, the basic structure of bR is the same in all cases). The s2 spectra468(the largest changes detected by the SVD analysis, whoseweightswere469about 7% (bR-NaClO4), 6% (bR-NaCl), and 7% (bR-NaCOOCH3) of their470corresponding s1 base spectrum) are also similar (Fig. 4B), meaning471that the temperature-induced spectral changes can be described by472the same phenomenology in all the three cases, namely !(II) (around4731667 cm!1) is the source structure of the temperature-induced474changes, and it is transformed into !(I) (1652 cm!1) [19,28] and475" (1622/1685 cm!1) [44,45] structures by increasing temperature.476(It should be noted that the s base spectra, in themselves, provide no477information about the sequence of these transitions.)478By the v2 vectors, the SVD analysis can reveal the temperature479dependence of the spectral changes. This is shown in Fig. 5, for the480Amide I" and II regions of Hofmeister salt-treated bR samples. The481increased values of the v2 vectors between 80 and 90 °C (Fig. 5)482re!ect the increased contribution of the corresponding s2 spectra to483the spectral changes, representing the transition from the original484!(II) structure to the product states, !(I) and ". In the Amide I"485band, the critical temperatures, where these large-scale structural486rearrangements start are 78 °C for bR-NaCOOCH3 (kosmotrope),48782 °C for bR-NaCl (HE-neutral), and 87 °C for bR-NaClO4 (chaotrope).488The critical temperatures (where the slow regime ends and the fast489one starts) were de"ned by the maxima of the second derivatives of490the v2(T) curves. (Note that these temperatures coincide well with491the declination temperatures in the !(II) curves in Fig. 3B.)492For the temperature dependence of the Amide II H/D exchange493(protein dynamics), the same sequence of Hofmeister salt effects494was found as for the transition temperatures of the Amide I" band.495In the Amide II v2 vectors, up to about 70–80 °C, there is a slow,496monotonously growing H/D exchange due to the increasing thermal497!uctuations in bR, and above 70–80 °C, a faster H/D exchange starts.498As discussed above, in the Amide II region only the disappearance of499the original peak takes place; therefore, the corresponding Amide II s2500base spectra were just negative bands centered at around 1550 cm!1,501and are not shown. (For details, see Figs. S3 and S4 in Supplementary502data.) The temperature dependence of the v2 vectors of the Amide II503region exhibits similar, albeit less dramatic, slope-changes as those of

Fig. 4. Singular value decomposition (SVD) of the Amide I" region of bR infrared spectrarecorded between 5 and 93 °C in different Hofmeister salts: NaClO4 — chaotropic, NaCl—neutral, and NaCOOCH3 — kosmotropic. A — The s1 base spectra of the SVD analysis,i.e., the average spectra over the temperature range studied. B — The s2 base spectra,i.e. the largest changes associatedwith the temperature-induced conformational changes.Note the negative band at around 1667 cm!1 assigned to !(II) helices, and the positivebands at around 1652, and 1622 cm!1, assigned to !(I) and " structures, respectively,into which the !(II) structures are transformed upon heat treatment.

Fig. 5. Temperature dependence of heat-induced structural changes as re!ected by thev2 vectors of the SVD analysis of the Amide I" (Am-I) and II (Am-II) regions of theinfrared absorption spectra of bR recorded in the presence of different Hofmeister salts.Numbers in corresponding colors indicate the critical temperatures of the unfolding ofthe original !(II) forms in the presence of different Hofmeister salts.

6 B. Szalontai et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

UNCO

RRECTED P

RO

OF

504 Amide I", but they start at 6–8 °C lower temperatures. It is remarkable505 that the slope of the bR-NaCOOCH3 (kosmotrope) curve in Fig. 5,506 below about 80 °C, is higher than that of the bR-NaClO4 (chaotrope).507 Above 80 °C, however, this relation reverses: the temperature depen-508 dence of the Amide II band disappearance (H/D exchange) is steeper509 in bR-NaClO4 than in bR-NaCOOCH3. (The HE-neutral bR-NaCl curve510 is in between the two extremes in both cases.) This means that511 under the critical temperature the chaotropic perchlorate, above it the512 kosmotropic acetate decreases the !exibility of bR.

513 4. Discussion

514 Comparison of the complementary information provided by the515 three different experimental methods is expected to give important516 clues for the interpretation of Hofmeister effects by conformational517 !uctuations, and, thereby, for understanding the interplay between518 surface properties and protein dynamics, too.519 The sequence and the character of the reactions the bR undergoes520 during temperature increase were revealed by FTIR and DSC mea-521 surements. FTIR experiments prove that the amount of the native522 !(II) elements in bR decreases dramatically in the 80–90 °C temper-523 ature range as evidenced by the dramatic decrease of 1667 cm!1

524 band in Fig. 3. The SVD analysis also shows here the increase of the525 contribution of the s2 base spectra (see the steep increase of the v2526 vectors in Fig. 5). In agreement with the prediction [11], the527 chaotropic Na-perchlorate stabilizes, while the kosmotropic Na-acetate528 destabilizes the native conformation as compared to the Hofmeister-529 neutral NaCl (see the critical temperatures in Fig. 5). The critical temper-530 atures assigned to the slope-brakes of the SVD v2 vectors of the Amide I"531 band (Fig. 5) seem to correlatewith the pre-transition peaks of the Cp(T)532 curves in the DSC measurements (Fig. 2). This correlation becomes533 understandable if we consider the following de"nition of heat capacity534 [27]:

Cp T# $ ! !T2%2G%T2

!: #4$

535536537 This means that the peaks of the Cp(T) curves are proportional to538 the curvature of the free energy function within a narrow tempera-539 ture range. This curvature is expected to be the highest at those540 temperatures where the most signi"cant acceleration of free energy541 changes takes place, due, e.g., to the onset of large-scale conforma-542 tional changes. From the FTIR measurements, on the other hand, the543 slope breaks of the v2(T) intensity functions of the second SVD base544 spectra (s2) mark just such points (Fig. 5), hence they should, in545 fact, well approximate the critical temperatures established from546 the Cp(T) curves (Fig. 2). (An exact coincidence cannot be expected,547 because v2(T) is not strictly proportional to G(T).)548 About conformational !uctuations, the neutron-scattering and the549 H–D exchange FTIR measurements provide the most direct informa-550 tion. They demonstrate that the intensity of !uctuations is in!uenced551 by Hofmeister salts in the entire investigated temperature range.552 When approaching the critical temperature of the !(II) ! !(I) phase553 transition, the salt-induced effects are getting enhanced (Figs. 1 and 5).554 The disappearance of the Amide II band of bR due to H/D exchange555 upon increasing temperatures shows the same order of critical tem-556 peratures for the three Hofmeister salts (CH3COO! b Cl! b ClO4

!) as557 observed for conformational changes via the Amide I' band (Fig. 5).558 Here, however, due to the rather smooth and broad transition from559 the low-rate H/D exchange to the high-rate one, it would be dif"cult560 to assign precise values to the critical temperatures. Nevertheless, it561 can be safely stated that the high-rate disappearance of the Amide II562 band of bR starts always at a few degrees lower temperature than563 that characteristic for the corresponding conformational changes as564 determined from the thermotropic responses of the Amide I" region

565and of Cp (Figs. 5 and 2, respectively). Since intra-molecular !uctua-566tions are needed to make the peptide \NH groups accessible for567the D+ ions, the increase of the H/D exchange rate already below568the critical temperatures determined from Cp(T) or v2(T) is indicative569of an increased rate of equilibrium !uctuation changes in the protein570preceding its conformational transition.571It is very interesting, how the Hofmeister salts affect these equilib-572rium !uctuations below and above the critical temperature. From the573FTIR experiments it turns out that below the Amide II critical temper-574atures, the kosmotropic NaCOOCH3 allows the steepest H/D exchange575in bR, i.e. it induces larger conformational !uctuations than the576HE-neutral NaCl or the chaotropic NaClO4 does (see the Am-II curves577in the low-temperature region of Fig. 5). Around the critical temper-578atures, large-scale structural rearrangements start, leading to protein579denaturing at the end. In the high-temperature region, the tempera-580ture dependence of the H/D exchange becomes steeper, too. Here,581evidently, larger segments of the bR molecules become at least tem-582porally accessible to the external D2O-rich medium on their way to583complete denaturing. Interestingly, for bR above the critical tempera-584ture, the !uctuation–Hofmeister salt relationship seems to reverse:585here, the slope of the H/D exchange curve becomes the steepest for bR586in the chaotropic perchlorate and the !attest for bR in the kosmotropic587acetate salt.588The above "ndings are in good agreement with the results of the589neutron scattering experiments, where the MSD(T) functions directly590measure !uctuations of the non-exchanged H atoms of amino acids,591that are supposedly buried in the innermost part of the protein. From592ca. 40 to 75 °C, the MSD values (characteristic of the !uctuations), as593well as the slopes of theMSD(T) curves (characteristic of the “!exibility”),594are higher for the kosmotrope-treated bR-NaCOOCH3 sample than for the595chaotrope-treated bR-NaClO4 one. On the other hand, the higher !exibil-596ity of bR-NaClO4 as compared to that of bR-NaCOOCH3 above the critical597temperature is apparently opposite to what was observed below this598temperature, similarly to the v2 vectors of the Amide II H/D exchange599data.600The temperatures corresponding to the change of the force con-601stant also seem to be in accordance with the slope-brakes in the602Amide II H/D exchange curves (Figs. 1 and 5). Since the Amide II H/D603exchange signal monitors more the outer residues (where the H/D604exchange has already taken place at a given temperature), this corre-605lation implies that the dynamical properties of the protein surface606determine the internal dynamics, too.607The onset of the !uctuation changes appears close to, but by a few608degrees lower than the critical temperatures found for Amide I" and609for the Cp peak, and alludes to a casual relationship between increased610intramolecular dynamics and the corresponding structural changes. A611similar phenomenon has been observed by neutron-scattering mea-612surements on bR at low temperatures [41], and has been interpreted613by the help of a double-well potentialmodel [42], showing that the !uc-614tuations start to increase at temperatures where the system “probes”615not only one, but both of the potential wells. This occurs already well616below the critical temperature of the transition [41,42].617In order to interpret the effects revealed by the present study, here618we also present a phenomenological free energy landscape model619based on the assumptions of Dér at al. [11], illustrating the energetics620of the temperature-induced conformational changes of bR in the621!(II) ! !(I) transition regime.622The free energy of the bacteriorhodopsin-solvent system is623depicted as a function of the solvent accessible surface area (ASA),624in Fig. 6. (Note that possible changes of the lipid–protein interface625are not considered in this model.) The two potential wells correspond626to the !(I) and !(II) conformations of bR, having smaller and larger627ASAs, respectively. The middle curve represents the situation for628bR-NaCl at its critical temperature (T = Tc(NaCl)). The equally629leveled bottoms of the potential minima have a positive or negative630slope corresponding to the more closed !(I) or to the more open

7B. Szalontai et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

UNCO

RRECTED P

RO

OF

631 !(II) conformations, respectively. (In this context, namely, an “open”632 conformation also implies that ASA tends to reach a local maximum,633 while in a “closed” conformation it “seeks after” a local minimum,634 within the borders of the free energy well.) Kosmotropic salts (like635 acetate) contribute with an additional linear background of a positive,636 while chaotropic salts (e.g., perchlorate) with an additional line of a637 negative slope to the landscape at the same temperature, due to the638 respective salt-induced increase or decrease of the protein–water639 interfacial tension (based on Gs = $pw · ASA). The model predicts sta-640 bilization of the !(II) conformation versus !(I) by chaotropic, and its641 destabilization by kosmotropic salts (&Gchao > 0, while &Gkosmo b 0).642 This relationship can also be quantitatively checked by the help of the643 experimental results. Namely, based on the &H and &Cp values for the644 !(II) ! !(I) transition determined from the calorimetric experiments,645 the corresponding &G values can be calculated at any given tempera-646 ture according to the Gibbs–Helmholtz equation:

&G ! &Hc 1!T=Tc# $!&Cp Tc!T# $ % T ln T=Tc# $& ' #5$

647648 where Tc is the critical temperature and &Hc is the enthalpy change of649 the transition up to Tc, while&Cp is the heat capacity difference between650 the !(I) and !(II) states. Eq. (5) assumes a temperature-independent651 approximation of&Cp. Since for bR in all the three salts&Cp is zerowithin652 the error, this condition is ful"lled in our case, and at the same time it653 allows a simple approximation of &Hc with &H/2. From the average654 experimental values of Table 1, at T = Tc(NaCl) = 82 °C (5) yields pos-655 itive (0.126 kcal·mol!1), zero and negative (!0.096 kcal·mol!1) &G656 values for bR-NaClO4, bR-NaCl and bR-NaCOOCH3, respectively, in agree-657 ment with the implications of the model.658 On the other hand, the model also predicts a lower level of confor-659 mational !uctuations for chaotropic salts (Wchao) as compared to660 kosmotropic ones (Wkosmo) below the phase transition, and an opposite661 relationship above it, giving a straightforward interpretation of the662 main experimental results of this study.

6635. Conclusions

664Using a complex experimental approach, we demonstrated that665Hofmeister-active anions affect the magnitude of protein conforma-666tional !uctuations in the entire temperature range studied, even in667the interior of the bacteriorhodopsin molecule. Chaotropic perchlo-668rate is found to decrease, and kosmotropic acetate to increase !uctu-669ations of the more open !(II) conformation, while they act vice versa670on the more closed !(I) form. We also found that, in each salt, an671onset of enhanced equilibrium !uctuations of the protein precedes672the temperature-induced!(II) ! !(I) phase transition. The obtained673results are in accordance with the predictions of theories outlined674earlier [11,42], and provide general implications to a better under-675standing of the governing principles of conformational !uctuations,676as essential attributes of protein dynamics and function [17].677Supplementary data to this article can be found online at http://678dx.doi.org/10.1016/j.bbagen.2013.05.036.

679Acknowledgements

680This research was sponsored by the Hungarian research grants:681KTIA-OTKA 78367 (A.D.), OTKA K-101821 (A.D.), K-75818 (B.Sz.),682K-101633 (T.P.) and CNK-80345, as well as by a bilateral grant between683the Hungarian and Bulgarian Academies of Sciences. We would like to684thank the Institut Laue–Langevin (Grenoble, France) for providing beam685time for the experiments. We also wish to thank Dr. Jérôme Combet for686the discussions concerning the neutron scattering data treatment and687Karine Huard for her help in sample preparation.

688References

689[1] F. Hofmeister, Zur Lehre von der Wirkung der Salze. II, Arch. Exp. Pathol.690Pharmacol. 24 (1888) 247–260.691[2] P. Lo Nostro, B.W. Ninham, Hofmeister phenomena: an update on ion speci"city692in biology, Chem. Rev. 112 (2012) 2286–2322.693[3] M.G. Cacace, E.M. Landau, J.J. Ramsden, The Hofmeister series: salt and solvent694effects on interfacial phenomena, Q. Rev. Biophys. 30 (1997) 241–277.695[4] K.D. Collins, M.W. Washabaugh, The Hofmeister effect and the behaviour of water696at interfaces, Q. Rev. Biophys. 18 (1985) 323–422.697[5] Q3P. Khoroshyy, A. Dér, L. Zimányi, Effect of Hofmeister cosolutes on the photocycle698of photoactive yellow protein at moderately alkaline pH, J. Photochem. Photobiol. B.699[6] C. Ganea, A. Babes, C. Lupfert, E. Grell, K. Fendler, R.J. Clarke, Hofmeister effects of700anions on the kinetics of partial reactions of the Na+, K+-ATPase, Biophys. J. 77701(1999) 267–281.702[7] R.M. Davidson, S. Seneff, The initial common pathway of in!ammation, disease,703and sudden death, Entropy 14 (2012) 1399–1442.704[8] W. Kunz, P. Lo Nostro, B.W. Ninham, The present state of affairs with Hofmeister705effects, Curr. Opin. Colloid Interface Sci. 9 (2004) 1–18.706[9] W. Melander, C. Horvath, Salt effects on hydrophobic interactions in precipitation707and chromatography of proteins — interpretation of lyotropic series, Arch.708Biochem. Biophys. 183 (1977) 200–215.709[10] M.C. Gurau, S.M. Lim, E.T. Castellana, F. Albertorio, S. Kataoka, P.S. Cremer, On the710mechanism of the Hofmeister effect, J. Am. Chem. Soc. 126 (2004) 10522–10523.711[11] A. Dér, L. Kelemen, L. Fábián, S.G. Taneva, E. Fodor, T. Páli, A. Cupane, M.G. Cacace,712J.J. Ramsden, Interfacial water structure controls protein conformation, J. Phys.713Chem. B 111 (2007) 5344–5350.714[12] T.J. Richmond, Solvent accessible surface area and excluded volume in proteins:715analytical equations for overlapping spheres and implications for the hydrophobic716effect, J. Mol. Biol. 178 (1984) 63–89.717[13] A. Dér, Salts, interfacial water and protein conformation, Biotechnol. Biotechnol.718Equip. 22 (2008) 629–633.719[14] Y. Zhang, P.S. Cremer, The inverse and direct Hofmeister series for lysozyme, Proc.720Natl. Acad. Sci. U. S. A. 106 (2010) 15249–15253.721[15] A. Neagu, M. Neagu, A. Dér, Fluctuations and the Hofmeister effect, Biophys. J. 81722(2001) 1285–1294.723[16] W. Stoeckenius, R.H. Lozier, R.A. Bogomolni, Bacteriorhodopsin and the purplemem-724brane of halobacteria, Biochim. Biophys. Acta Rev. Bioenerg. 505 (1979) 215–278.725[17] A. Dér, J.J. Ramsden, Evidence for loosening of a protein mechanism,726Naturwissenschaften 85 (1998) 353–355.727[18] S. Krimm, A.M. Dwivedi, Infrared spectrum of the purple membrane: clue to a728proton conduction mechanism? Science 216 (1982) 407–708.729[19] J. Wang, M.A. El-Sayed, The effect of protein conformation change from !(II) to730!(I) on the bacteriorhodopsin photocycle, Biophys. J. 78 (2000) 2031–2036.731[20] S.M. Barnett, C.M. Edwards, I.S. Butler, I.W. Levin, Pressure-induced transmembrane732!II- to !I-helical conversion in bacteriorhodopsin: an infrared spectroscopic study,733J. Phys. Chem. B 101 (1997) 9421–9424.

Fig. 6. Double-well model of the free energy landscape of bacteriorhodopsin at the!(II) ! !(I) transition temperature of bR-NaCl (T = Tc(NaCl)). Gibbs solute-waterfree energy (Gs) of bR is plotted as a function of the solvent accessible surface area(ASA) in various salt solutions. The more closed secondary structure element (withsmaller ASAs) is !(I), while the more open one (with bigger ASAs) is !(II). The middlecurve (green) represents undisturbed landscape of the Hofmeister-neutral bR-NaCl.The upper curve (blue) is the landscape of the kosmotropic bR-acetate, distorted byadding an increasing linear baseline re!ecting the increased protein–water interfacialtension caused by the presence of the kosmotropic acetate (NACOOCH3) salt [11].The lower curve (red) was obtained by adding a negative background to the originallandscape to show the effect of the decreased protein–water interfacial tension, dueto the effect of the chaotropic perchlorate [11]. The salt-dependence of the magnitudeof the ASA !uctuations (W) and the free energy differences between the !(I) and !(II)states (&G) are shown by the arrow-headed lines. kT is the depth of the Boltzmann“energy !uid”. Indices kosmo, neut, chao, refer to kosmotropic, neutral, and chaotropicHofmeister salts, respectively.

8 B. Szalontai et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036

UNCO

RRECTED P

RO

OF

734 [21] D. Oesterhelt, W. Stoeckenius, Isolation of the cell membrane of Halobacterium735 halobium and its fractionation into red and purple membrane, Methods Enzymol.736 31 (1974) 667–678.737 [22] M. Bée, Quasielastic Neutron Scattering: Principles and Applications in Solid State738 Chemistry, Biology, and Materials Science, Adam Hilger, Bristol, England and739 Philadelphia, 1988.740 [23] F. Gabel, D. Bicout, U. Lehnert, M. Tehei, M. Weik, G. Zaccai, Protein dynamics741 studied by neutron scattering, Q. Rev. Biophys. 35 (2002) 327–367.742 [24] J.C. Smith, Protein dynamics: comparison of simulations with inelastic neutron743 scattering experiments, Q. Rev. Biophys. 24 (1991) 227–291.744 [25] H.H. Paalman, C.J. Pings, Numerical evaluation of X-ray absorption factors for745 cylindrical samples and annular sample cells, J. Appl. Phys. 33 (1962) 2635–2639.746 [26] D. Richard, M. Ferrand, G.J. Kearley, Analysis and visualisation of neutron-747 scattering data, J. Neutron Res. 4 (1996) 33–39.748 [27] N.V. Prabhu, K.A. Sharp, Heat capacity in proteins, Annual Review of Physical749 Chemistry, 2005, pp. 521–548.750 [28] S.G. Taneva, J.M.M. Caaveiro, A. Muga, F.M. Goni, A pathway for the thermal751 destabilization of bacteriorhodopsin, FEBS Lett. 367 (1995) 297–300.752 [29] Z.A. Tokaji, E.A.B. Fodor, A.A. Szabó-Nagy, T.A. Páli, Hydroxylamine as a thermal753 destabiliser of bacteriorhodopsin, Eur. Biophys. J. 39 (2010) 1605–1611.754 [30] C. Zscherp, A. Barth, Reaction-induced infrared difference spectroscopy for the755 study of protein reaction mechanisms, Biochemistry 40 (2001) 1875–1883.756 [31] P. Zavodszky, J.T. Johansen, A. Hvidt, Hydrogen-exchange study of conformational757 stability of human carbonic-anhydrase-B and its metallocomplexes, Eur. J. Biochem.758 56 (1975) 67–72.759 [32] E. Goormaghtigh, V. Cabiaux, J.M. Ruysschaert, Determination of soluble and mem-760 brane protein structure by Fourier transform infrared spectroscopy. II. Experimental761 aspects, side chain structure, and H/D exchange, Subcell. Biochem. 23 (1994) 363–403.762 [33] E.R. Henry, J. Hofrichter, Singular value decomposition: application to analysis of763 experimental data, Methods Enzymol. 210 (1992) 129–192.764 [34] H.A. Laczkó-Dobos, B.b Szalontai, Lipids, proteins, and their interplay in the765 dynamics of temperature-stressed membranes of a cyanobacterium, Synechocystis766 PCC 6803, Biochemistry 48 (2009) 10120–10128.

767[35] K. Wood, U. Lehnert, B. Kessler, G. Zaccai, D. Oesterhelt, Hydration dependence of768active core !uctuations in bacteriorhodopsin, Biophys. J. 95 (2008) 194–202.769[36] G. Zaccai, Moist and soft, dry and stiff: a review of neutron experiments on770hydration-dynamics–activity relations in the purple membrane of Halobacterium771salinarum, Biophys. Chem. 86 (2000) 249–257.772[37] U. Lehnert, V. Réat, M. Weik, G. Zaccaï, C. P"ster, Thermal motions in bacteriorho-773dopsin at different hydration levels studied by neutron scattering: correlation774with kinetics and light-induced conformational changes, Biophys. J. 75 (1998)7751945–1952.776[38] M. Ferrand, A.J. Dianoux, W. Petry, G. Zaccai, Thermal motions and function of777bacteriorhodopsin in purple membranes — effects of temperature and hydration778studied by neutron scattering, Proc. Natl. Acad. Sci. U. S. A. 90 (1993)7799668–9672.780[39] J. Fitter, R.E. Lechner, G. Buldt, N.A. Dencher, Internal molecular motions of bacte-781riorhodopsin: hydration-induced !exibility studied by quasielastic incoherent782neutron scattering using oriented purple membranes, Proc. Natl. Acad. Sci. U. S. A.78393 (1996) 7600–7605.784[40] H.B. Callen, T.A. Welton, Irreversibility and generalized noise, Phys. Rev. 83785(1951) 34–40.786[41] D.J. Bicout, G. Zaccai, Protein !exibility from the dynamical transition: a force787constant analysis, Biophys. J. 80 (2001) 1115–1123.788[42] G.A.B. Zaccai, How soft is a protein? A protein dynamics force constant measured789by neutron scattering, Science 288 (2000) 1604–1607.790[43] I. Jelesarov, H.R. Bosshard, Isothermal titration calorimetry and differential scanning791calorimetry as complementary tools to investigate the energetics of biomolecular792recognition, J. Mol. Recognit. 12 (1999) 3–18.793[44] J.L. Arrondo, A.Muga, J. Castresana, F.M. Goni, Quantitative studies of the structure of794proteins in solution by Fourier-transform infrared spectroscopy, Prog. Biophys. Mol.795Biol. 59 (1993) 23–56.796[45] I.H. van Stokkum, H. Linsdell, J.M. Hadden, P.I. Haris, D. Chapman, M. Bloemendal,797Temperature-induced changes in protein structures studied by Fourier trans-798form infrared spectroscopy and global analysis, Biochemistry 34 (1995)79910508–10518.800

801

9B. Szalontai et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Please cite this article as: B. Szalontai, et al., Hofmeister ions control protein dynamics, Biochimica et Biophysica Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.05.036