Hofmeister ions control protein dynamics

This theory, implying a “liquid drop model”, predicts changes in protein conformational fluctuations upon addition of Hofmeister salts containing either kosmotropic or chaotropic anions t

Hofmeister ions control protein dynamics

Balázs Szalontaia, Gergely Nagyb,c,1, Sashka Krumovad, Elfrieda Fodore, Tibor Pália, Stefka G Tanevad,2, Győző Garabf, Judith Petersc,3, András Déra,⁎

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

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

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

d Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad G Bonchev Str., Bl.21, 1113 Sofia, Bulgaria

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

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

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 16 April 2013

Received in revised form 24 May 2013

Accepted 28 May 2013

Available online 7 June 2013

Keywords:

Hofmeister effect

Differential scanning calorimetry

Neutron scattering

Fourier transform infrared spectroscopy

Protein structural fluctuation

Bacteriorhodopsin

Background: Recently, we have elaborated a thermodynamic theory that could coherently interpret the diverse effects of Hofmeister ions on proteins, based on a single physical parameter, the protein–water interfacial tension (Dér et al., Journal of Physical Chemistry B 2007, 111, 5344–5350) This theory, implying a “liquid drop model”, predicts changes in protein conformational fluctuations upon addition of Hofmeister salts (containing either kosmotropic or chaotropic anions) to the medium

Methods: Here, we report experimental tests of this prediction using a complex approach by applying methods especially suited for the detection of protein fluctuation changes (neutron scattering, micro-calorimetry, and Fourier-transform infrared spectroscopy)

Results: It is demonstrated that Hofmeister salts, via setting the hydrophobic/hydrophilic properties of the pro-tein–water interface, control conformational fluctuations even in the interior of the typical membrane transport protein bacteriorhodopsin, around its temperature-induced, unusual α(II) → α(I) conformational transition between 60 and 90 °C We found that below this transition kosmotropic (COOCH3

−

), while above it chaotropic (ClO4

−

) anions increase structural fluctuations of bR It was also shown that, in each case, an onset of enhanced equilibrium fluctuations presages this phase transition in the course of the thermotropic response of bR Conclusions: These results are in full agreement with the theory, and demonstrate that predictions based on protein–water interfacial tension changes can describe Hofmeister effects and interpret protein dynamics phenom-ena even in unusual cases

General significance: This approach is expected to provide a useful guide to understand the principles governing the interplay between protein interfacial properties and conformational dynamics, in general

© 2013 Elsevier B.V All rights reserved

1 Introduction

Hofmeister effects (HEs) cover a wide range of salt-induced

phe-nomena on proteins, and on colloidal particles in general, including

changes of solubility, denaturation, or enzyme kinetics In his original

work, Hofmeister [1] reported that certain salts (primarily their anions) either decrease or increase protein solubility when present

in the solution at moderate or high concentrations (usually above

100 mM), and arranged them into the so-called Hofmeister series (HS) according to the sign and the magnitude of the effect they exerted The

HS of the most important anions, in descending order of their precipitat-ing ability, is as follows: F−≈ SO42−> HPO42−> CH3COO−> Cl−>

NO3−> Br−

> ClO3−> I−

> ClO4−> SCN−

Members in the series left

of the “Hofmeister-neutral” Cl−

are precipitants, while the ones right

of it are solubilizers An impressive number of follow-up studies show that the same HS emerges in other phenomena, like denaturation of proteins, inhibition or activation of enzymes, as well (for reviews, see

[2–4]) HEs live their renaissance in the past few years Besides their significance in colloid chemistry, preparative biochemistry and biotech-nology, they have been successfully applied in studying the function and dynamics of macromolecular structures[5,6] In addition, a recent review calls the attention to their role in basic pathophysiologic issues

[7]

⁎ Corresponding author at: Institute of Biophysics, Biological Research Centre, Hungarian

Academy 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, 5232

Villigen PSI, Switzerland.

2 Present address: Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de

Bioquímica y Biología Molecular, Universidad del País Vasco, 48080 Bilbao, Spain.

3 Present address: University Joseph Fourier, UFR PhITEM, BP 53, 38041 Grenoble

Cédex 9, France; Institut de Biologie Structurale, 41 rue Jules Horowitz, 38027 Grenoble

Cédex 1, France.

0304-4165/$ – see front matter © 2013 Elsevier B.V All rights reserved.

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Despite the widespread occurrence of HEs, and the extensive

research efforts focused on them, their interpretation has remained

a matter of debate[8], because of the lack of a unifying formalism

covering the entire spectrum of salts from “salting out” to “salting

in” effects, i.e from precipitants to solubilizers One approach[9]

cor-related these attributes with effects on water structure, in particular

on the fraction of hydrogen-bonded water molecules: precipitants

promote H-bond formation between the water molecules in their

vicinity, and are therefore called kosmotropes, while solubilizers

break H-bonds between water molecules, and are therefore called

chaotropes In another branch of interpretations, dispersion forces

were suspected to be the main factor responsible for HEs [10]

Although these are certainly among the main microphysical factors

responsible for the Hofmeister effects, all attempts aiming to assign

a single physical parameter as an approximate measure for the

whole diversity of HEs, however, have failed for more than a century

In a recent work, we have demonstrated that a unified,

phenome-nological formalism is able to qualitatively account for the entire

range of HE-related phenomena[11] The crucial difference between

our approach and the previous attempts of similar intention was

that, instead of building on the conventionally used air–water

inter-face[9], we took protein–water interfacial tension (γpw) as the

prin-cipal physical parameter to describe Hofmeister effects The most

important conclusion of our theory is that HEs are manifested via

the surface-dependent term of the free energy (Gs= γpw· ASA,

where ASA is the solvent accessible surface area) of the proteins

(or, in general, of colloid disperse systems) [12] In other words,

they modify the hydrophobic effect at protein–water interfaces[13]

The addition of kosmotropic or chaotropic salts to the solvent

increases or decreases the solute–water interfacial tension,

respec-tively, and thus, Gs should increase or decrease accordingly The

alteration of Gs is the driving force behind the observed HEs

affecting either the aggregation or the conformation of the proteins

Note that γpwshould, necessarily, depend also on the quality of the

solvent-exposed protein surface, and may even take negative values,

too (e.g., in case of proteins of naturally open conformation)[11,14]

An implication of our theory is that HEs are manifested by

chang-ing transition rates and equilibrium constants in reactions

accompa-nying major conformational changes that involve changes in the

water-exposed surface area of macromolecules and supramolecular

assemblies (Such effects have recently been exemplified by a

de-tailed study of Hofmeister effects on the function of photoactive

yellow protein[5].) It was also established that interfacial tension and

protein stability are interconnected by protein conformational

fluctua-tions, thus providing the keystone for the microscopic interpretation

of HEs[11,15] In consequence of the above view, the same HS should

appear for salt-induced protein conformational fluctuations as for

pre-cipitation or conformational changes

In spite of the success of this theory in describing the diverse

Hofmeister phenomena, so far direct experimental evidences for

salt-induced changes of protein conformational fluctuations have

been missing Hence, here we used a complex experimental approach

to monitor temperature-induced changes of protein structure and

dynamics, with methods specially adapted for observing changes in

conformational fluctuations (neutron scattering, differential scanning

calorimetry (DSC), and Fourier transform infrared spectroscopy

(FTIR)) Our model object was the prototypical retinal protein,

bacte-riorhodopsin (bR) from Halobacterium salinarum[16] Also known

as the simplest ion pump in biological systems, bR is one of the

best-characterized and most robust membrane proteins, subjected

to non-specific anion binding effects Despite, both its structure and

function have been shown to be influenced by interactions with

Hofmeister anions [11,17] In the present study, special attention

was paid to the reversible conformational change of bR in the course

of its heat denaturation, the α(II) → α(I) conformational transition

between 60 and 90 °C, where enhanced changes of structural

fluctuations were expected to arise [18,19], due to the associated alterations of the solvent-exposed surface

So far, there have been only a few reports about proteins having

an “open” conformation as their naturally most stable form[3], and

bR is considered to be such one Namely, previous FTIR measurements

on bR detected α(II) helices below ca 50 °C[18], and between 60 and

90 °C, bR undergoes an α(II) → α(I) conformational transition[19]

In α(I), however, the helix is known to be more tightly packed than

in α(II)[18,20] It implies that, in contrast to most proteins, here the accessible surface area (ASA) of the protein is higher at lower temperatures (α(II)), and ASA decreases upon the temperature-induced transition of the secondary structure (to α(I)) For such a case, our theory [11] predicts an unusual phenomenon to occur: instead of kosmotropic, here chaotropic salts should have a stabilizing effect; consequently, they are expected to shift the α(II) → α(I) tran-sition to higher temperatures, while kosmotropes are expected to destabilize the open α(II) protein conformation, therefore shifting the transition toward lower temperatures, as it has been indicated

by our former DSC experiments[11] We took advantage of this unusual feature of bR to test the predictive power of our HE-interpreting theory

2 Materials and methods 2.1 Bacteriorhodopsin-containing purple membrane (bR) isolation Purple membranes were isolated according to the standard proce-dure[21] Purple membranes were always re-suspended in D2O-based buffers, due to the needs of neutron scattering and FTIR experiments (10 mM HEPES in D2O at pD 6.6, containing 500 mM kosmotropic, Hofmeister-neutral, or chaotropic salts, NaCOOCH3, NaCl, or NaClO4, respectively, as required) The bR samples, treated with these salts will be denoted as bR-NaCOOCH3, bR-NaCl, and bR-NaClO4throughout the paper

2.2 Neutron scattering experiments For neutron scattering experiments the suspensions of bR-containing purple membranes were centrifuged for 20 min at 40,000 g The pellets were placed in flat rectangular aluminum sample holder with an area (30 × 40 mm2) adapted to the dimensions of the incident neutron beam The sample holders were hermetically closed and used for the experiment

The experiments were carried out on the IN13 backscattering spectrometer at the Institut Laue Langevin (ILL, France) Two samples were measured by elastic incoherent neutron scattering to determine the atomic mean-square displacements bu2> as a function of temper-ature One of the samples was bR-NaClO4, measured in the 40–91 °C temperature range, the other one was bR-NaCOOCH3measured be-tween 40 and 87 °C Temperature scans were done upon increasing the temperature in steps of 7–10 °C below the transition region, and

of 3–4 °C around the transition region Additional vanadium, buffer and empty cell measurements were recorded for correction and nor-malization purposes[22]

In the examined time range (8 μeV energy resolution, correspond-ing to a time window of 100 ps through Heisenberg's uncertainty principle) on backscattering spectrometers, H-atom motions reflect the motions of the chemical groups to which they are bound[23] The scattered elastic incoherent intensity can be described within the Gaussian approximation[24]by

IelðQ ; ϖ ¼ 0  ΔEÞ∝I0exp −1

2

Q2

ð1Þ

where I0is the intensity hitting the sample, bu2> is the average atomic mean square displacement, ω corresponds to the energy transfer, and Q

to the momentum transfer between neutron and target The average

value of the atomic mean square displacements (MSD) for the protein

can thus be obtained for each temperature from the slope of the

semi-logarithmic plot of the incoherent scattering function through

u2

¼ −6d lnIelðQ ; ϖ ¼ 0  ΔEÞ

The data treatment, including subtraction of the scattering from

the empty sample holder, normalization to vanadium and absorption

correction based on the correction formula of Paalman–Pings

coeffi-cients[25]was performed using the ILL program LAMP (Large Array

Manipulation Program[26])

2.3 Differential scanning calorimetry experiments

Differential scanning calorimetry (DSC) is a useful tool to

charac-terize the thermodynamics of protein conformational changes[27],

and has been applied to monitor the steps of thermal denaturation

of bR, too[11,28,29] Direct information about the specific heat of

the protein (Cp) can be derived from the experiments, and Cp is

instantaneously related to enthalpy fluctuations, according to the

fluctuation-dissipation theorem[27]:

Cp¼b δH

2

>

DSC measurements were performed on bR membrane suspensions of

6 mg·mL−1protein concentrations, by using a VP-DSC high-sensitivity

differential scanning calorimeter (MicroCal Inc., Northampton, MA)

equipped with built-in twin reference and sample cells of 0.5 mL

volume Temperature scans were performed in the 10–110 °C range, at

a heating rate of 0.5 °C·min−1, and the differential heat capacities

were recorded At the end of the heating scans samples were cooled

back to 10 °C where they were allowed to equilibrate for 30 min before

being subjected to a second heating scan with the same measuring

pro-tocol These re-heating scans served later as baselines for the first

record-ings since they did not show any transition peak present in the above

mentioned temperature range Positions of the pre- and main transition

peaks were determined by using the built-in integration routine of the

DSC analysis software (MicroCal-Origin, DSC application)

2.4 Infrared measurements

FTIR spectra were recorded on a Philips PU9800 Fourier transform

infrared spectrometer, averaging 128 scans at 2 cm− 1spectral

resolu-tions For the infrared experiments, 1.5 mL aliquots of bR-containing

purple membranes dispersed in the required D2O-based buffer +

Hofmeister salt, were concentrated in a Hettich (Germany) EBA 12R

tabletop centrifuge (4 °C, 24,000 ×g, 8 min) The pellet (about 20 μL)

was placed between CaF2windows separated by an aluminum spacer

(15 μm) Temperature dependence of the infrared absorption spectra

was measured by recording 35 × 2 FTIR spectra between 5 and 95 °C

in about 3 °C steps, leaving 7 min for reaching thermal equilibrium At

each temperature, two absorption spectra were measured by recording

for each single beam background (empty spectrometer) and sample

spectrum (CaF2window plus purple membrane dispersion) by using

a sample shuttle A water-thermostated cell holder controlled the

tem-perature of the sample The accuracy of the temtem-perature setting was

about 0.1 °C

In water suspensions of most biological membranes, the Amide I

band is centered at around 1650 cm− 1, arising mainly from the

C_O stretching vibration (with minor contributions from the

out-of-phase C\N stretching vibration, the C\C\N deformation

and the N\H in-plane bend) The Amide II mode near 1550 cm− 1is

the out-of-phase combination of the NH in-plane bend and the CN

stretching vibration with smaller contributions from CC, NC stretching vibrations, and CO in-plane bend[30]

To keep the Amide I region free from the disturbing strong contri-bution of the H2O-bending band at 1643 cm− 1, and being in agree-ment with the neutron scattering and DSC experiagree-ments, all infrared spectra were recorded in D2O environment The presence of D2O induces the exchange of the peptide \NH groups to \ND ones, which affects the Amide I and II regions differently The H/D isotope effect is weak in the Amide I region, since N\H vibrations have little contribution to these modes, as discussed above (the shape of the Amide I region does not change considerably, only the frequencies

of the component bands shift down by a few wavenumbers; the deuterated Amide I band will be indicated throughout the paper as Amide I′) Temperature-dependent changes in the Amide I′ remain characteristic to the changes of the secondary structure elements of the protein

To monitor protein dynamics, we used the Amide II region, where H/D exchange exerts a major effect, since the Amide II region down-shifts to about 1450 cm− 1(Amide II′) Therefore, the Amide II band (around 1550 cm− 1) disappears upon H/D exchange The rate of this disappearance is informative about protein structural fluctuations

[31,32], since the access of the external D atoms to the peptide \NH groups depends on the protein dynamics under the given environ-mental factors (temperature, molecular interactions)

2.4.1 Singular value decomposition (SVD) analysis Temperature-dependent changes in the infrared spectra were studied by SVD analysis[33] Briefly, the D (n ∗ m) data matrix was decomposed as D = S ∗ W ∗ VT, where n is the number of wave-lengths, and m is the number of temperatures at which the measure-ments were made S (n ∗ m) contains the orthonormal abstract spectrum vectors; the W (m ∗ m) diagonal matrix consists of the singular values in descending order, while V (m ∗ m) contains the orthonormal vectors describing the temperature dependence The s1base spectrum (the first column of the S matrix) shows the average spectrum over the temperature range involved; s2shows the largest changes accompanying the temperature increase, which have

to be combined with the s1spectrum to get back the actual spectrum

at a given temperature The v1and v2amplitude vectors give the temperature dependence of the average, and the largest change, respectively, manifested in the s1, s2spectra, etc In the present SVD analysis, we considered only the average (s1, v1) and the largest change (s2, v2) of the spectra recorded upon increasing temperature (5–95 °C) The higher order SVD components (s3, v3, s4, v4…, etc.) were neglected because their weights were low This approach is evidently an approximation, which, however, can be justified since the weights (wi) of the sibase spectra were decreasing very rapidly (to about 2–3% of w1by w3, data not shown)

All calculations were carried out with the SPSERV© software of Cs Bagyinka (Biological Research Centre, Szeged, Hungary) For more details on the use of SVD analysis on biological membrane proteins, see our earlier work on dynamics and temperature-induced denatur-ation of proteins in thylakoid membranes[34]

3 Results 3.1 Neutron scattering experiments Among the three applied methods, neutron scattering gives the most direct information about conformational fluctuations It probes mainly the mean square displacements (MSD) of hydrogen atoms in biological samples, since the incoherent cross-section of H is much larger than that of any other atom in the samples Since H atoms are usually homogeneously distributed in biological samples, the MSD are assumed to reflect the global dynamics of the system as well

The bR-containing purple membranes consist of two types of

molecules, bR and the lipid components of the membranes, therefore,

in principle, we should see in the scattering experiments

contribu-tions from both lipids and bR Nevertheless, by choosing the

temper-ature range where bR, but not the lipid, is expected to have a major

structural transition, and taking into account the 75:25 weight ratio

of bR to lipids in purple membranes, one can assume that the changes

seen in H-dynamics are mostly due to H-atoms of bR, and that

lipid-related H atoms provide, at most, a smoothly changing

back-ground A further factor to be taken into account is the H/D exchange

at the \NH groups of the solvent accessible peptide bonds This

means that especially at higher temperatures, the decreasing

popula-tion of non-D2O-accessible peptide groups, which are either in the

interior or at the lipid interface of bR, will have the major contribution

to the neutron scattering signals, i.e., this method monitors the least

accessible, internal parts of the protein

Bacteriorhodopsin dynamics has been studied by incoherent

neu-tron scattering over the past decades[35–39] In these experiments,

two populations of motions have been found to arise when

investigat-ing the thermotropic behavior of fully hydrated bR with neutron

scat-tering They were assigned to dynamical transitions around −123 °C

and −23 °C, where onsets of additional conformational motions occur

[37] In the present case, however, we measured at much higher

temper-atures (in the 40–91 °C range), in order to monitor Hofmeister effects on

protein fluctuations associated with the α(II) → α(I) transition of bR

Neutron scattering experiments were carried out on bR-containing

purple membranes suspended in D2O-based kosmotropic acetate and

chaotropic perchlorate salt solutions (500 mM NaCOOCH3and NaClO4,

respectively)

InFig 1, MSD values of hydrogen atoms are plotted as a function

of temperature They are associated with the sample flexibility via the

fluctuation-dissipation theorem[40] At higher temperatures, where

the solvent of the protein is fluid, the elastically scattered intensity

decreases drastically, as the motions are leaving the instrumental time

window This fact leads to high errors (compare our errors bars with

those in Ref.[41]) of the calculated MSD values Nevertheless, a break in

the slope of the MSD(T) function for kosmotrope-treated bR-NaCOOCH3 around 75 °C, and for the chaotrope-treated bR-NaClO4around 80 °C is clearly visible The mean force constants, which are associated with the stability of the whole sample, can be separately calculated for the two temperature regions according to the kh i ¼ 0:00276

d u h 2 i=dTformula[36,42] The fitted straight lines and their slopes of the obtained MSD values and the force constants derived from their slopes are shown inFig 1 Note, that the chaotropic sample below the transition has a higher mean force con-stant (a lower slope of the fitted curve) associated with a lower flexibility

as compared to that of the kosmotropic sample Above the transition, however, the calculated 〈k〉 is slightly lower for the chaotropic sample than for the kosmotropic one, but the difference is within the error of the measurements In other words, below the transition the resistance against structural fluctuations is higher (i.e., the conformational flexibility

is lower) in bR-NaClO4than in bR-NaCOOCH3, while above the transition, they are about the same in the two cases (or might even be oppositely related) This implies a higher “dynamical stability” of bR-NaClO4than that of bR-NaCOOCH3, being also reflected by the up-shift of the transition temperature of bR by the chaotropic salt

3.2 Calorimetric measurements

In order to correlate fluctuation changes with thermodynamic transitions, complementary differential scanning calorimetry (DSC) experiments were performed under similar ambient conditions (i.e., in

D2O-based media) For the DSC measurements, however, the set of salt solutions was extended by the Hofmeister-neutral chloride, in order to allow a full comparison with results obtained earlier with H2O-based salts[11], too

Fig 2shows that the main unfolding transition peak of the Cp(T) curve is located at around 100 °C, slightly depending on the particular Hofmeister salt, while a minor peak (the so-called “pre-transition”)

Fig 1 MSDs as the function of temperature for bR-containing purple membranes

suspended in D 2 O-based 0.5 M kosmotropic (NaCOOCH 3 ) and chaotropic (NaClO 4 )

Hofmeister salt solutions Straight lines represent linear fits used to extract the force

constants bk> A change in the slope of the MSD values can be observed at around

75 °C for bR-NaCOOCH 3 , and at around 80 °C for bR-NaClO 4 The lower conformational

flexibility below the transition and the higher transition temperature in bR-NaClO 4

compared to bR-NaCOOCH 3 suggest a higher dynamical stability for the chaotrope-treated

Fig 2 DSC heating endotherms of bR-containing purple membranes suspended in

D 2 O-based solutions of 0.5 M of various Hofmeister salts Temperature profiles of excess molar heat capacities from top to bottom correspond to samples in the presence

of chaotropic NaClO 4 (red), neutral NaCl (green), and kosmotropic NaCOOCH 3 (blue), respectively The two vertical lines indicate the temperature range, in which the α(II) → α(I) transition of bR takes place in the various Hofmeister salts The numbers indicate the critical temperatures (Tc) associated to the reversible α(II) → α(I) transi-tion (“pre-transitransi-tion”) and the irreversible (denaturing) maxima The scanning rate was 0.5 °C·min −1 The data were subjected to baseline subtraction by using the second heating scans as baselines, and were normalized to the corresponding protein concen-trations For better visibility, the NaClO 4 and NaCOOCH 3 curves were up- and downshifted,

appears in the 75-90 °C range In all cases, transitions could not be

detected in the second heating scans, indicating that during the first

scan a complete and irreversible heat-denaturation of the protein

occurred This indicates that the main transition peak of the Cp(T)

curve most probably reflects the final, irreversible denaturation step

of the bR accompanied also by a decomposition of the purple

mem-brane Therefore, in the following we do not consider the analysis of

this peak From our point of view, much more interesting is the

minor, reversible pre-transition in the 75–90 °C range, which exhibits

strong Hofmeister salt dependence This transition has been assigned

to the α(II) → α(I) conformational change[19,28] The chaotropic

ClO4−

ions shifted the critical temperature (Tc) of this transition up

to ca 88 °C, as compared to the Hofmeister-neutral Cl−

around

82 °C, while the kosmotropic CH3COO−ions downshifted the same

transition to about 77 °C It is worth mentioning that these values are

by a few degrees higher than the ones measured on non-deuterated

bR[11], and that the peaks related to the α(II) → α(I) transitions are

getting sharper when moving from the kosmotropic acetate to the

chaotropic perchlorate (Fig 2)

From the Cp(T) curves, the molar enthalpy and heat capacity changes

(ΔH and ΔCp values, respectively) associated with the α(II) → α(I)

transition were also determined according to Jelesarov and Bosshard

[43]for bR suspended in the three different salt solutions (Table 1)

The molar enthalpy changes were determined from the integral of

the Cp(T) functions, and found to be the same for the three different

salts within the error, implying that the main differences between

the initial and product secondary structures of the protein (α(II) and

α(I) states, respectively) are presumably the same in all the three

cases The corresponding ΔCpvalues, determined from lines fitted to

the Cp(T) functions before and after the transition, are close to zero

within the temperature range of interest

3.3 Fourier transform infrared (FTIR) experiments

To reveal the sequence and the ion dependence of the secondary

structure changes in bR, FTIR experiments were carried out on different

Hofmeister salt-treated bR samples The salt content of the FTIR samples

was the same as those of used in the neutron scattering and DSC

mea-surements The Amide I′ and Amide II regions of the infrared spectra,

characteristic of the secondary structure and dynamics of proteins,

were recorded and analyzed

The sequence of the secondary structure changes (from α(II)

through α(I) to β), could be examined by the temperature

depen-dence of the evolution of the related infrared absorption bands in

the difference spectra, obtained by subtracting the absorption

spec-trum recorded at the lowest temperature from the later ones Since

all the three samples behaved qualitatively similar (see Fig S1 in

Supplementary data) we present, as an example, the evolution of

changes in the Amide I′ region upon temperature increase only for

the kosmotrope-treated bR-NaCOOCH3 (Fig 3) (The same results

for the other two salts are given in Fig S2.) In the difference spectra

depicted here (Fig 3A), first, a shallow valley around 1667 cm− 1is

the only feature, which slowly deepens toward higher temperatures

In agreement with the literature, this frequency was assigned to

α(II) helices[19,28] Around 80–85 °C, the changes become more

characteristic, partly due to the deepening of the 1667 cm− 1band,

and partly due to an emerging band at around 1652 cm− 1, assigned

to α(I) helices [44] Above 90 °C, the strong, β-structure-related band at around 1622 cm−1 dominates the difference spectra (one may observe a weaker pair-band around 1685 cm−1as well inFig 3A

as it should be[44,45]) This pair of bands was assigned earlier to anti-parallel β-sheets[44] The appearance of this type of β-structure always accompanies the heat-induced denaturation of proteins[45] For better visualizing the sequence of events, we have plotted just the maxima of the three bands as a function of temperature inFig 3B FromFig 3one may conclude that on the way to heat-induced denatur-ation of bR, first the α(II) structure is transformed to α(I), then α(I) (and the remaining α(II)) to β-sheets The same sequence of events could be established for bR-NaCl, and bR-NaClO4, as well, except that the corresponding spectral changes started at higher temperatures (shown in Fig S2)

Table 1

Molar enthalpy and heat capacity changes during the temperature-induced α(II) → α(I)

transition of bacteriorhodopsin, and the corresponding critical absolute temperatures.

bR-NaOOCH 3 bR-NaCl bR-NaClO 4

ΔH (kcal·mol−1) 11.4 ± 0.5 9.7 ± 3.1 10.9 ± 0.4

ΔC p (kcal·mol −1 ·K −1 ) 0.4 ± 0.5 − 0.5 ± 0.5 − 0.7 ± 0.5

T c (°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 presence

of 0.5 M NaCOOCH 3 (kosmotropic) Hofmeister salt upon increasing temperatures.

A — Difference absorption spectra obtained by subtracting the first infrared absorption recorded 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 panel

A Note that first α(I) starts to grow at the expense of α(II), then both α-structures diminish into the emerging β-band, which is indicative of irreversible protein denaturation.

In order to give a more rigorous analysis of the similarities in the

secondary structures of bR in the presence of different Hofmeister

salts, and of the differences between the dynamics and stability of

these structures upon heat-induced changes, we analyzed the

tem-perature series of the recorded infrared spectra with SVD (Fig 4)

(The advantage of this pure mathematical procedure is that it does

not require any model for the decomposition of the measured spectra,

and uses the whole data set on the full spectral and temperature

range investigated[33].) The s1and s2base spectra provide

informa-tion about the spectral changes of the samples in the investigated

temperature range

Fig 4A shows the s1base spectra (corresponding to the average

structure of the temperature series) derived from the Amide I′

regions of bR in the presence of different Hofmeister salts recorded

between 5 and 93 °C It can be seen that s1spectra are very similar,

i.e the majority of the secondary structure elements contributing to

the Amide I′ region are not affected by the Hofmeister salts (in other words, the basic structure of bR is the same in all cases) The s2spectra (the largest changes detected by the SVD analysis, whose weights were about 7% (bR-NaClO4), 6% (bR-NaCl), and 7% (bR-NaCOOCH3) of their corresponding s1base spectrum) are also similar (Fig 4B), meaning that the temperature-induced spectral changes can be described by the same phenomenology in all the three cases, namely α(II) (around

1667 cm− 1) is the source structure of the temperature-induced changes, and it is transformed into α(I) (1652 cm− 1) [19,28] and

β (1622/1685 cm− 1) [44,45] structures by increasing temperature (It should be noted that the s base spectra, in themselves, provide no information about the sequence of these transitions.)

By the v2vectors, the SVD analysis can reveal the temperature dependence of the spectral changes This is shown inFig 5, for the Amide I′ and II regions of Hofmeister salt-treated bR samples The increased values of the v2 vectors between 80 and 90 °C (Fig 5) reflect the increased contribution of the corresponding s2spectra to the spectral changes, representing the transition from the original α(II) structure to the product states, α(I) and β In the Amide I′ band, the critical temperatures, where these large-scale structural rearrangements start are 78 °C for bR-NaCOOCH3 (kosmotrope),

82 °C for bR-NaCl (HE-neutral), and 87 °C for bR-NaClO4(chaotrope) The critical temperatures (where the slow regime ends and the fast one starts) were defined by the maxima of the second derivatives of the v2(T) curves (Note that these temperatures coincide well with the declination temperatures in the α(II) curves inFig 3B.)

For the temperature dependence of the Amide II H/D exchange (protein dynamics), the same sequence of Hofmeister salt effects was found as for the transition temperatures of the Amide I′ band

In the Amide II v2 vectors, up to about 70–80 °C, there is a slow, monotonously growing H/D exchange due to the increasing thermal fluctuations in bR, and above 70–80 °C, a faster H/D exchange starts

As discussed above, in the Amide II region only the disappearance of the original peak takes place; therefore, the corresponding Amide II s2 base spectra were just negative bands centered at around 1550 cm−1, and are not shown (For details, see Figs S3 and S4 in Supplementary data.) The temperature dependence of the v2vectors of the Amide II region exhibits similar, albeit less dramatic, slope-changes as those of

Fig 4 Singular value decomposition (SVD) of the Amide I′ region of bR infrared spectra

recorded between 5 and 93 °C in different Hofmeister salts: NaClO 4 — chaotropic, NaCl —

neutral, and NaCOOCH3— kosmotropic A — The s 1 base spectra of the SVD analysis,

i.e., the average spectra over the temperature range studied B — The s 2 base spectra,

i.e the largest changes associated with the temperature-induced conformational changes.

Note the negative band at around 1667 cm −1 assigned to α(II) helices, and the positive

bands at around 1652, and 1622 cm −1 , assigned to α(I) and β structures, respectively,

Fig 5 Temperature dependence of heat-induced structural changes as reflected by the

v 2 vectors of the SVD analysis of the Amide I′ (Am-I) and II (Am-II) regions of the infrared absorption spectra of bR recorded in the presence of different Hofmeister salts Numbers in corresponding colors indicate the critical temperatures of the unfolding of

Amide I′, but they start at 6–8 °C lower temperatures It is remarkable

that the slope of the bR-NaCOOCH3 (kosmotrope) curve in Fig 5,

below about 80 °C, is higher than that of the bR-NaClO4(chaotrope)

Above 80 °C, however, this relation reverses: the temperature

depen-dence of the Amide II band disappearance (H/D exchange) is steeper

in bR-NaClO4than in bR-NaCOOCH3 (The HE-neutral bR-NaCl curve

is in between the two extremes in both cases.) This means that

under the critical temperature the chaotropic perchlorate, above it the

kosmotropic acetate decreases the flexibility of bR

4 Discussion

Comparison of the complementary information provided by the

three different experimental methods is expected to give important

clues for the interpretation of Hofmeister effects by conformational

fluctuations, and, thereby, for understanding the interplay between

surface properties and protein dynamics, too

The sequence and the character of the reactions the bR undergoes

during temperature increase were revealed by FTIR and DSC

mea-surements FTIR experiments prove that the amount of the native

α(II) elements in bR decreases dramatically in the 80–90 °C

temper-ature range as evidenced by the dramatic decrease of 1667 cm− 1

band inFig 3 The SVD analysis also shows here the increase of the

contribution of the s2base spectra (see the steep increase of the v2

vectors in Fig 5) In agreement with the prediction [11], the

chaotropic Na-perchlorate stabilizes, while the kosmotropic Na-acetate

destabilizes the native conformation as compared to the

Hofmeister-neutral NaCl (see the critical temperatures inFig 5) The critical

temper-atures assigned to the slope-brakes of the SVD v2vectors of the Amide I′

band (Fig 5) seem to correlate with the pre-transition peaks of the Cp(T)

curves in the DSC measurements (Fig 2) This correlation becomes

understandable if we consider the following definition of heat capacity

[27]:

Cpð Þ ¼ −TT 2 ∂

2G

∂T2

!

This means that the peaks of the Cp(T) curves are proportional to

the curvature of the free energy function within a narrow

tempera-ture range This curvatempera-ture is expected to be the highest at those

temperatures where the most significant acceleration of free energy

changes takes place, due, e.g., to the onset of large-scale

conforma-tional changes From the FTIR measurements, on the other hand, the

slope breaks of the v2(T) intensity functions of the second SVD base

spectra (s2) mark just such points (Fig 5), hence they should, in

fact, well approximate the critical temperatures established from

the Cp(T) curves (Fig 2) (An exact coincidence cannot be expected,

because v2(T) is not strictly proportional to G(T).)

About conformational fluctuations, the neutron-scattering and the

H–D exchange FTIR measurements provide the most direct

informa-tion They demonstrate that the intensity of fluctuations is influenced

by Hofmeister salts in the entire investigated temperature range

When approaching the critical temperature of the α(II) → α(I) phase

transition, the salt-induced effects are getting enhanced (Figs 1 and 5)

The disappearance of the Amide II band of bR due to H/D exchange

upon increasing temperatures shows the same order of critical

tem-peratures for the three Hofmeister salts (CH3COO−

b Cl−b ClO4−) as observed for conformational changes via the Amide I' band (Fig 5)

Here, however, due to the rather smooth and broad transition from

the low-rate H/D exchange to the high-rate one, it would be difficult

to assign precise values to the critical temperatures Nevertheless, it

can be safely stated that the high-rate disappearance of the Amide II

band of bR starts always at a few degrees lower temperature than

that characteristic for the corresponding conformational changes as

determined from the thermotropic responses of the Amide I′ region

and of Cp(Figs 5 and 2, respectively) Since intra-molecular fluctua-tions are needed to make the peptide \NH groups accessible for the D+ions, the increase of the H/D exchange rate already below the critical temperatures determined from Cp(T) or v2(T) is indicative

of an increased rate of equilibrium fluctuation changes in the protein preceding its conformational transition

It is very interesting, how the Hofmeister salts affect these equilib-rium fluctuations below and above the critical temperature From the FTIR experiments it turns out that below the Amide II critical temper-atures, the kosmotropic NaCOOCH3allows the steepest H/D exchange

in bR, i.e it induces larger conformational fluctuations than the HE-neutral NaCl or the chaotropic NaClO4does (see the Am-II curves

in the low-temperature region ofFig 5) Around the critical temper-atures, large-scale structural rearrangements start, leading to protein denaturing at the end In the high-temperature region, the tempera-ture dependence of the H/D exchange becomes steeper, too Here, evidently, larger segments of the bR molecules become at least tem-porally accessible to the external D2O-rich medium on their way to complete denaturing Interestingly, for bR above the critical tempera-ture, the fluctuation–Hofmeister salt relationship seems to reverse: here, the slope of the H/D exchange curve becomes the steepest for bR

in the chaotropic perchlorate and the flattest for bR in the kosmotropic acetate salt

The above findings are in good agreement with the results of the neutron scattering experiments, where the MSD(T) functions directly measure fluctuations of the non-exchanged H atoms of amino acids, that are supposedly buried in the innermost part of the protein From

ca 40 to 75 °C, the MSD values (characteristic of the fluctuations), as well as the slopes of the MSD(T) curves (characteristic of the “flexibility”), are higher for the kosmotrope-treated bR-NaCOOCH3sample than for the chaotrope-treated bR-NaClO4one On the other hand, the higher flexibil-ity of bR-NaClO4as compared to that of bR-NaCOOCH3above the critical temperature is apparently opposite to what was observed below this temperature, similarly to the v2vectors of the Amide II H/D exchange data

The temperatures corresponding to the change of the force con-stant also seem to be in accordance with the slope-brakes in the Amide II H/D exchange curves (Figs 1 and 5) Since the Amide II H/D exchange signal monitors more the outer residues (where the H/D exchange has already taken place at a given temperature), this corre-lation implies that the dynamical properties of the protein surface determine the internal dynamics, too

The onset of the fluctuation changes appears close to, but by a few degrees lower than the critical temperatures found for Amide I′ and for the Cppeak, and alludes to a casual relationship between increased intramolecular dynamics and the corresponding structural changes A similar phenomenon has been observed by neutron-scattering mea-surements on bR at low temperatures[41], and has been interpreted

by the help of a double-well potential model[42], showing that the fluc-tuations start to increase at temperatures where the system “probes” not only one, but both of the potential wells This occurs already well below the critical temperature of the transition[41,42]

In order to interpret the effects revealed by the present study, here

we also present a phenomenological free energy landscape model based on the assumptions of Dér at al.[11], illustrating the energetics

of the temperature-induced conformational changes of bR in the α(II) → α(I) transition regime

The free energy of the bacteriorhodopsin-solvent system is depicted as a function of the solvent accessible surface area (ASA),

inFig 6 (Note that possible changes of the lipid–protein interface are not considered in this model.) The two potential wells correspond

to the α(I) and α(II) conformations of bR, having smaller and larger ASAs, respectively The middle curve represents the situation for bR-NaCl at its critical temperature (T = Tc(NaCl)) The equally leveled bottoms of the potential minima have a positive or negative slope corresponding to the more closed α(I) or to the more open

α(II) conformations, respectively (In this context, namely, an “open”

conformation also implies that ASA tends to reach a local maximum,

while in a “closed” conformation it “seeks after” a local minimum,

within the borders of the free energy well.) Kosmotropic salts (like

acetate) contribute with an additional linear background of a positive,

while chaotropic salts (e.g., perchlorate) with an additional line of a

negative slope to the landscape at the same temperature, due to the

respective salt-induced increase or decrease of the protein–water

interfacial tension (based on Gs= γpw· ASA) The model predicts

sta-bilization of the α(II) conformation versus α(I) by chaotropic, and its

destabilization by kosmotropic salts (ΔGchao> 0, while ΔGkosmob 0)

This relationship can also be quantitatively checked by the help of the

experimental results Namely, based on the ΔH and ΔCpvalues for the

α(II) → α(I) transition determined from the calorimetric experiments,

the corresponding ΔG values can be calculated at any given

tempera-ture according to the Gibbs–Helmholtz equation:

ΔG¼ ΔHcð1−T=TcÞ−ΔCp½ðTc−TÞ þ T ln T=Tð cފ ð5Þ

where Tcis the critical temperature and ΔHcis the enthalpy change of

the transition up to Tc, while ΔCpis the heat capacity difference between

the α(I) and α(II) states Eq.(5)assumes a temperature-independent

approximation of ΔCp Since for bR in all the three salts ΔCpis zero within

the error, this condition is fulfilled in our case, and at the same time it

allows a simple approximation of ΔHcwith ΔH/2 From the average

experimental values ofTable 1, at T = Tc(NaCl) = 82 °C(5)yields

pos-itive (0.126 kcal·mol−1), zero and negative (−0.096 kcal·mol−1) ΔG

values for bR-NaClO4, bR-NaCl and bR-NaCOOCH3, respectively, in

agree-ment with the implications of the model

On the other hand, the model also predicts a lower level of

confor-mational fluctuations for chaotropic salts (Wchao) as compared to

kosmotropic ones (Wkosmo) below the phase transition, and an opposite

relationship above it, giving a straightforward interpretation of the

main experimental results of this study

5 Conclusions Using a complex experimental approach, we demonstrated that Hofmeister-active anions affect the magnitude of protein conforma-tional fluctuations in the entire temperature range studied, even in the interior of the bacteriorhodopsin molecule Chaotropic perchlo-rate is found to decrease, and kosmotropic acetate to increase fluctu-ations of the more open α(II) conformation, while they act vice versa

on the more closed α(I) form We also found that, in each salt, an onset of enhanced equilibrium fluctuations of the protein precedes the temperature-induced α(II) → α(I) phase transition The obtained results are in accordance with the predictions of theories outlined earlier[11,42], and provide general implications to a better under-standing of the governing principles of conformational fluctuations,

as essential attributes of protein dynamics and function[17] Supplementary data to this article can be found online athttp:// dx.doi.org/10.1016/j.bbagen.2013.05.036

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

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