Volume properties and spectroscopy: A terahertz Raman investigation of hen eggwhite lysozymePaola Sassi, Stefania Perticaroli, Lucia Comez, Alessandra Giugliarelli, Marco Paolantoni, Daniele Fioretto, and

Assunta Morresi Citation: The Journal of Chemical Physics 139, 225101 (2013); doi: 10.1063/1.4838355 View online: http://dx.doi.org/10.1063/1.4838355 View Table of Contents: http://scitation.aip.org/content/aip/journal/jcp/139/22?ver=pdfcov Published by the AIP Publishing

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THE JOURNAL OF CHEMICAL PHYSICS 139, 225101 (2013)

Volume properties and spectroscopy: A terahertz Raman investigation ofhen egg white lysozyme

Paola Sassi,1,a) Stefania Perticaroli,2 Lucia Comez,3,4 Alessandra Giugliarelli,1

Marco Paolantoni,1 Daniele Fioretto,4,5 and Assunta Morresi11Dipartimento di Chimica, Università di Perugia, Via Elce di Sotto, 8, I-06123 Perugia, Italy2Chemical and Materials Sciences Division at Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831,USA and Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, USA3IOM-CNR c/o Dipartimento di Fisica, Università di Perugia, Via Pascoli, I-06123, Perugia, Italy4Dipartimento di Fisica, Università di Perugia, Via Pascoli, I-06123 Perugia, Italy5Centro di Eccellenza sui Materiali Innovativi Nanostrutturati (CEMIN), Università di Perugia, via Elce diSotto 8, 06123 Perugia, Italy

(Received 4 September 2013; accepted 19 November 2013; published online 9 December 2013)

The low frequency depolarized Raman spectra of 100 mg/ml aqueous solutions of hen egg whitelysozyme (HEWL) have been collected in the 25–85 ◦C range. Short and long exposures to hightemperatures have been used to modulate the competition between the thermally induced reversibleand irreversible denaturation processes. A peculiar temperature evolution of spectra is evidencedunder prolonged exposure of the protein solution at temperatures higher than 65 ◦C. This result isconnected to the self-assembling of polypeptide chains and testifies the sensitivity of the technique tothe properties of both protein molecule and its surrounding. Solvent free spectra have been obtainedafter subtraction of elastic and solvent components and assigned to a genuine vibrational contributionof hydrated HEWL. A straight similarity is observed between the solvent-free THz Raman featureand the vibrational density of states as obtained by molecular dynamics simulations; according tothis, we verify the relation between this spectroscopic observable and the effective protein volume,and distinguish the properties of this latter respect to those of the hydration shell in the pre-meltingregion. © 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4838355]

I. INTRODUCTION

The stability of the native state of a globular proteinis the result of a complex balance between different intra-and intermolecular interactions; any alteration of such a del-icate equilibrium is at the origin of its degradation routes.1–3

Nonetheless, the native state of the protein has not a rigid ar-rangement as local fluctuations are allowed and surely relatedto the mechanism by which the protein fulfills its activity.Surprisingly, in spite of the obvious biological interest, littleattention4–6 has been devoted to the characterization of pos-sible gradual conformational changes in the pre-melting tem-perature region with respect to studies of unfolding transition.

The main differences between properties of the proteinstructure in these two temperature domains (pre-melting andmelting) clearly appeared in a recent work in which some ofus have investigated the role of hydration in the structure andthermostability of lysozyme in water-glycerol mixtures, bymeans of photon correlation spectroscopy (PCS) and circulardichroism.7 For the average protein radius Rh as a function ofincreasing temperature over the range 20–85 ◦C, PCS exper-iment evidenced two distinct behaviors: a relative maximumof Rh at T = 40–50 ◦C followed by a second major change atunfolding.

a)Author to whom correspondence should be addressed. Electronicmail: paola.sassi@unipg.it. Telephone: +39 075 5855585. Fax:+39 075 5855586.

In the temperature interval 5–55 ◦C dielectric measure-ments at radiofrequencies on lysozyme aqueous solution werealso performed8 thus verifying a continuous gradual changeof the hydrodynamic radius with a maximum in the pre-melting region, in agreement with PCS data.

In the pre-melting region we also observed a changeof temperature dependence for the position of amide I IRabsorption.9 Due to the relation between this spectral featureand the secondary structure of the polypeptide chain, we sug-gested a local conformational change to occur in the 40–50 ◦Crange.

In this article, the low frequency Raman spectra of100 mg/ml aqueous solutions of HEWL in the 25–85 ◦C rangeare presented and discussed. Two different thermal treatmentshave been applied to protein solutions to distinguish betweeneffects of the unfolding of the single molecule (reversibledenaturation) and those of self-assembling (irreversible de-naturation). In fact, protein aggregation is kinetically drivenand the resulting irreversible denaturation is only evidencedat long thermalization times unless an organic co-solvent isadded to the solution.9–12 We recently used IR absorption,Raman and Brillouin scattering spectroscopies to characterizethe structure of both single molecule and supramolecular ar-rangements in the same samples studied in the present work.13

Under the long thermalization treatment we observed thatclustering occurs at high temperatures with a slow kinetics.13

The increase of longitudinal viscosity was assessed by thesharp variation of position and width of Brillouin peaks at

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225101-2 Sassi et al. J. Chem. Phys. 139, 225101 (2013)

75 ◦C, where, concomitantly, a thermally induced gelationwas also revealed. The secondary structure of HEWL in thegel state was characterized by the analysis of amide I bandof Raman and IR spectra: an unfolded arrangement of sin-gle chains and the lack of ordered supramolecular structureswere observed. Hence, under reversible and irreversible ther-mal denaturation we evidenced the same secondary structureof the protein molecule; moreover, the same tertiary structurewas also revealed in the two samples by following S–S andside-chain marker bands of Raman spectra.

Recently, a number of experimental reports and compu-tational analyses concerning the low-frequency response ofproteins systems14–17 have evidenced the characteristic fea-tures of this spectral region. The low frequency motions mustarise from collective vibrations extending to the whole proteinmolecule, or very large portions of it;18–22 unfortunately, therelation between this spectral feature and the protein structure(whether it exists) is still not definite.23 Moreover, most ofattempts to discuss about this spectral region refer to the to-tal profile due to vibrational and relaxation properties of bothwater and protein; on the contrary we largely demonstratedthat Extended frequency range Depolarized Light Scattering(EDLS) technique can be used to separate the effects of thesetwo entities.24, 25

The goals of the present work are manifold. On oneside, we show the sensitivity of Raman susceptibility to re-veal the different environments of protein chains under re-versible and irreversible denaturation. Moreover, we confirmthe volume expansion discussed in Ref. 7 (PCS data); to thisextent, we perform density measurements to compare withlow-frequency Raman results. Finally, due to this comparison,we evidence the relation between the THz spectroscopic ob-servable and the protein volume, and particularly the effectivecontribution of HEWL molecule with respect to its hydrationshell.

II. MATERIALS AND METHODS

A. Materials

HEWL (purity, 90%) was purchased from Sigma-Aldrichand used without further purification. Lysozyme molecule has129 amino acid residues and a molecular weight of 14.3 kDawith an isoelectric point of 10.7 and an ellipsoidal shapecharacterized by semiaxes 1.9 × 2.2 × 1.1 nm. A 100 mglysozyme/ml water solution was prepared by weight, addingthe protein to doubly distilled deionized H2O; the completedissolution of the biomolecule was achieved by heating at40 ◦C for ∼1 h, and the pH value (∼4) was controlled with aFiveEasy pH meter FE20 Mettler Toledo. Maximum catalyticactivity in lysozyme is observed around this pH value.26 pHconditions were also checked during the heating process anda +0.4 units variation was observed at higher temperatures.The solution was filtered with Millipore filters of 0.20 μm di-ameter pores directly in the measure cuvette in order to obtaindust free samples. Reproducibility of data was checked by re-peating both measurements and data manipulation on threefresh samples.

B. Thermal treatment

Two different thermal treatments were applied to HEWLsolutions according to procedure reported in Ref. 13. In sam-ple 1 (S1) a 2 ◦C/min rate and 30 min thermalization at 25.0,35.0, 50.0, 65.0, 67.5, 70.0, 72.5, 75.0, 80.0, 82.5, and 85.0 ◦Cwere used upon heating the solution. In sample 2 (S2) theheating run was performed with the same rate but with muchlonger thermalization times: 4 h for each temperature value(slow heating). Data were replicated three times on a freshsolution in order to test the reproducibility of results.

C. Raman spectra

Depolarized light scattering intensities (IHV) wererecorded between 1 and 30 cm−1 with a resolution of0.4 cm−1 and at 4–1200 cm−1 with a resolution of 1 cm−1.Spectra were acquired using a Coherent-Innova 90 Ar+ laseroperating on a single mode at λ = 514.5 nm line (typicalpower of 300 mW) as a light source, and a Jobin-Yvon modelU1000 double monochromator having 1 m focal length withholographic gratings. The scattered light was detected us-ing a thermoelectrically cooled (−30 ◦C) Hamamatsu model943XX photomultiplier. The temperature on the sample wascontrolled by circulating water from a Haake F6 ultrather-mostat with a precision of 0.1 ◦C. Low and high frequencyspectra were spliced, exploiting an overlap of about threedecades in wavenumbers. Subsequently, the imaginary partof the dynamic susceptibility χ ′′(ν) was calculated accordingto the relation χ ′′(ν) = IHV(ν)/[nB(ν) + 1], where nB(ν) = 1/[exp(hν/kBT) − 1] is the Bose-Einstein occupation number.

D. Density measurements

Density measurements in the range 20–65 ◦C were per-formed with a Paar Model DMA512 densimeter connected toa vibrating Paar Model DMA60 cell; the values are given withan error of 0.5%.

III. RESULTS AND DISCUSSION

A. Solvent-free χ ′′ profiles

In Figure 1(a) the spectra of neat water are shown as afunction of temperature in the range 25–85 ◦C; Figure 1(b)shows the spectra of S1 in the same range of temperature. Allχ ′′ profiles were rescaled on the intensity of the librationalband (300–1000 cm−1) of pure water at 25 ◦C; this band isscarcely affected by concentration and thermal variations inthis temperature range.

From the comparison of Figures 1(a) and 1(b) it is ob-served that profiles of a 100 mg/ml HEWL solution are alwaysmuch more intense than those of water, suggesting a prepon-derant contribution of the hydrated solute in this spectral re-gion. Moreover, since the intensity of water profile seems tobe scarcely affected by thermal variations, also the markedenhancement observed for the solution at temperatures higherthan 70 ◦C is essentially ascribed to the protein.

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FIG. 1. THz Raman susceptibility of water (a) and a 100 mg/ml HEWL so-lution (b) in the 25–85 ◦C temperature range. THz Raman spectra are shownin a log (main figures) and linear (insets) wavenumber scale. We remark thatthe inset to each figure is a more detailed representation (enlarged ordinatescale) of profiles.

The lowest frequency portion of each χ ′′ spectrum re-veals an intense scattering due to the Tyndall effect and re-flecting the instrumental slit function; this latter was measuredon a latex solution and subtracted from S1 and S2 spectra. Torationalize the intensity enhancement of χ ′′ spectra localizedbetween 2 and 300 cm−1, we also removed the solvent contri-bution after rescaling on water librations.25 The resulting pro-file, hereafter called solvent free (SF) spectrum, is assignedto a mere vibrational contribution of lysozyme in solution.In the solution spectra the two low-frequency bands of waterat 50 and 180 cm−1 are overlapped to this vibrational feature.Our subtraction procedure implicitly assumes that these waterbands are temperature sensitive but pretty independent on thepresence of HEWL at 100 mg/ml concentration; this was veri-fied in one of our previous works by using EDLS technique.25

It is worth noting that, in that case, a more extended frequencyrange was analyzed since we were particularly interested tocharacterize the relaxation processes of bulk and hydrationwater. In this work we rather focus on the vibrational prop-erties of HEWL and experimental data have been obtainedat a lower resolution thus impeding to probe these relaxationfeatures at v̄ < 2 cm−1. In Ref. 25 we tested the subtractionprocedure by comparing EDLS data with results of inelas-

FIG. 2. SF profiles of S1(a) and S2(b) as a function of temperature.

tic neutron scattering27 and OHD-RIKES19 experiments, andby curve-fit analysis of the entire (0.02–1200 cm−1) spectraldomain. In particular, the 2–300 cm−1 SF distribution of S1sample at 25–70 ◦C (native state) was analyzed in terms ofmultiple Brownian oscillators thus giving fitting parameterperfectly consistent with OHD-RIKES study,19 and scarcelydependent on temperature; this supported the assignment tovibrational motions of hydrated HEWL.

In Figure 2 SF spectra evaluated in the 2–300 cm–1 regionare shown for both S1 and S2 as a function of temperature. Asa general trend, a significant intensity variation is observed athigher temperatures; the augment of intensity is much higherfor S1.

The spectral feature of Figure 2 extends over a three hun-dreds wavenumbers with an asymmetric shape sloping downon the high frequency side. Due to this asymmetric distribu-tion, an average frequency value is referred to the first mo-ment M1 of the band:

M1 =∫band

v̄I (v̄)dv̄∫band

I (v̄)dv̄. (1)

To account for the complete distribution of vibrationalstates we integrated the 2–300 cm−1 range of SF profiles; in-tegrated intensities together with M1 values as a function oftemperature, are displayed in Figures 3(a) and 3(b), respec-tively. The same pretty constant value of integrated intensityis measured for SF spectra of S1 and S2 at T = 25–70 ◦C;

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FIG. 3. Integrated intensity (a) and first moment (b) of S1 and S2 SF profiles.

on the contrary, a significant intensity variation is observed athigher temperatures and particularly for S1 sample. Actually,this intensity rise could be due to the activation of new vi-brational motions, to the amplitude increase of chain motionsor to the drastic change of potential wells surrounding thesecollective modes. In any case this is not a simple temperatureeffect but is rather connected to the global conformational re-arrangement taking place at melting. In fact, the melting ofboth secondary and tertiary structure of HEWL in water so-lution is realized in the 70–80 ◦C range (melting temperatureTm = 74 ◦C10).

It is worth noting the straight similarity between profilesof Figure 2 and the vibrational densities of states (VDOS) ofHEWL in water solution as recently obtained by room tem-perature MD simulations.14 Actually, this similarity has beenobserved in many disordered systems28–30 and Raman sus-ceptibility in the THz regime is commonly referred as de-scriptive of VDOS, but the total contribution of both soluteand solvent is usually addressed. On the contrary, our pro-files allow us to isolate the inelastic response of the protein insolution.

MD simulations by van Vlijmen and Karplus31 indicatedthat the overall change of protein structure occurring in thetransition range causes an increase of atomic fluctuations ofthe partially and totally unfolded forms of HEWL: this caninduce much larger polarizability changes and then a higherscattering intensity. The larger atomic fluctuations of proteinunits testify a larger flexibility of polypeptide chains: this islikely connected with the partial loss of secondary and tertiary

structures due to the lessening of intramolecular interactions.The comparison of data in Figure 3(a) shows that this increaseof flexibility caused by the thermal unfolding of the singlemolecule is reduced due to the strengthening of intermolec-ular contacts that causes cluster formation. Thus, the depo-larized light scattering shows a great sensitivity in revealingeffects caused by changing of both intra- and intermolecularinteractions.

Together with a higher mobility of vibrating units, a newdistribution of collective vibrational modes and a higher den-sity of states for frequencies <15 cm−1 are observed for thenon-native states.31 This effect is at the origin of the augmentof χ ′′ intensity and also the downshift of the average value ofcollective vibrational frequencies.

Our experimental data evidence a downshift of aver-age oscillating frequency in the whole 20–70 ◦C range (seeFigure 3(b)). In particular, a sharp decrease of M1 value isobserved at 60 < T < 70 ◦C, at the beginning of the melt-ing process. This suggests an abrupt lessening of intramolecu-lar interactions respect to the room-temperature conformationand the softening of the structure leading to unfolding. Due tothe fact that S1 and S2 have the same secondary and tertiarystructure at high and low temperatures,13 the same M1 valuesare obtained from analysis of corresponding SF profiles in thewhole temperature range.

The sudden weakening of intramolecular interactions atthe beginning of the main transition might seem in contrastwith Rh data of Figure 4; in fact, in this temperature rangethese data evidence a shrinking of the hydrodynamic vol-ume. Rh values are indicative of the apparent size of pro-tein molecule and were calculated from the estimate of thediffusion coefficient of HEWL in solution obtained by PCSexperiments.7 Actually, the presence of a maximum in thetemperature evolution of Rh could result from the competi-tion of two opposite effects. The initial growth of the hydro-dynamic radius could be attributed to the thermal expansionof the tertiary structure of the protein as a result of increasedfluctuations of the native state. Leading to the unfolding of

FIG. 4. Apparent specific volume VLys and hydrodynamic radius Rh respec-tively obtained from density measurements and photon correlation spectra(Rh values are reproduced from Ref. 7). In the inset, the thermal expan-sion coefficient of HEWL native state as obtained from average collectivefrequency (αM1) and densitometry (αdens) are shown.

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225101-5 Sassi et al. J. Chem. Phys. 139, 225101 (2013)

the chain, this expansion should be particularly effective whenapproaching the transition; on the contrary, one observes thedecline of Rh. A collapse of the hydration shell of HEWLin this range was derived from analysis of results of wide-angle X-ray scattering measurements.32 Actually, the changeof hydrating conditions can be connected to the decrease ofhydrodynamics radius since this quantity is related to the vol-ume of solvating layer together with the effective volume ofthe protein. Unfortunately, Rh data were obtained at slightlydifferent solvating conditions: presence of a small glycerolfraction and a lower pH.7 Thus, possible differences in vol-ume effects could be ascribed to the different expansion in-duced by the environment on both folded and unfolded stateof HEWL. As a consequence, in order to test a possible con-nection between the low frequency vibrations and the volu-metric properties of the protein, we performed density mea-surements on S1 sample and compared these data with thetemperature dependence of M1 in the native state. The com-parison was restricted to the native state of S1 in order toconsider only the temperature effects on a definite (folded)structure.

B. Thermal expansion of the native state

The apparent specific volume of the protein VLys (cm3

g−1) can be calculated from the measured values of the so-lution and solvent density, ρ and ρ0 respectively, using thefollowing relation:33–35

VLys = 1

ρ0·(

1 − ρ − ρ0

wρ

), (2)

where w is the weight fraction of protein in solution.Volume changes accompanying the melting of globular

proteins have been explored to investigate the solvation prop-erties of these systems, and the few available studies of therecent literature suggest a common behavior, with a thermalexpansivity of the folded structure that is decreasing on in-creasing temperature.34–36 Usually, highly diluted conditions(w < 0.02) are analyzed to evaluate the partial specific vol-ume of a macromolecule and characterize its hydration shellduring the heat-induced or chemically-induced denaturation.The HEWL concentration of our sample (w = 0.1) couldcause a non-negligible contribution of protein-protein inter-actions to be considered on estimation of apparent volume.Nonetheless, the room temperature value of VLys we assessedby densitometric method is in perfect agreement with infinitedilution data;34 on the contrary, the temperature evolution ofVLys here presented is much more pronounced than the oneobserved in diluted solution at pH = 2.5.34, 36 This is probablydue to the different pH value of present work being sensitivelyhigher (pH ∼ 4) than the diluted case. In fact, it has been ob-served that reducing the acidity of the solution (and thus thepositive charge on the protein surface) causes a softening ofthe structure and favors the thermal expansion of the nativestate.33

In Figure 4 the temperature dependence of VLys showsa maximum value at about 50 ◦C, in agreement with Rh be-havior reported in Ref. 7 and shown in the same Figure 4. Rhvalues were obtained for HEWL in water-glycerol mixtures

while we assessed the apparent specific volume VLys in aque-ous solution; surprisingly, a quite good agreement betweenthese two sets of results is evidenced by direct comparison.Actually, from VLys we evaluated the protein radius RV byconsidering the HEWL molecule as a spherical particle, andobtained that RV is smaller than Rh (16.2 instead of 20.0 Åat 35 ◦C), but, particularly, its temperature variation between50 and 65 ◦C is much less pronounced (0.5 instead of 5%).This can be connected to the different solvating conditionsexploited in Ref. 7 respect to our sample, but, most of all,we believe it can be due to the different sensitivity of the twotechniques to probe the volume of the protein and its tem-perature variations. Anyway, density measurements definitelyconfirm the expansion of the native state from room temper-ature up to 50 ◦C, and the decrease of apparent volume at thebeginning of the main transition.

In a recent paper by Xu et al.37 the low-lying vibrationalmodes of the native BSA were modeled as those of a threedimensional, elastic nanoparticle with aperiodic charge distri-bution. According to a solid-like description of the nanopar-ticle, we refer to the low-frequency vibrations of HEWLas phonon modes of a disordered solid. The normal modephonon frequencies of a quasi-harmonic solid decrease withincreasing its volume V (v̄phonon∝ V−γ ),38 such that the rela-tive changes of v̄phonon with T can be applied to calculate thevolume expansion coefficient αM1 of the sample according toEq. (3):

αM1 = − 1

γ · ν̄phonon· ∂v̄phonon

∂T. (3)

The parameter γ is the Grüneiser parameter; it is usuallyassumed equal to unity thus giving for this protein a α value of∼10−3 K−1, very close to those obtained from inelastic neu-tron scattering of bacteriorhodopsin.39 The thermal expansioncoefficient of HEWL native state can be also evaluated fromthe relative change of apparent specific volume as a functionof temperature according to Eq. (4):

αdens = 1

VLys

· ∂VLys

∂T. (4)

In the inset of Figure 4 the comparison between αM1 andαdens values shows the perfect agreement between the twomethods of evaluating the expansivity of the native state andsupport the assignment of the low frequency modes describedby SF spectra to phonon-like vibrations of HEWL molecule.A different description of the THz feature of HEWL spectrumcould refer to the contribution of local vibrational modes ex-tending to portions of the protein structure: also in this case,a change of protein volume would affect the strength of in-teractions experienced by different residues and cause a shiftof vibrational frequencies.17 Therefore, one can reasonablyassume that M1, as a probe of intramolecular interactions de-termining the folded structure of the protein, has the sametemperature dependence of the inverse of molecular volume.

C. Protein volume in the pre-melting region

In the range 20–50 ◦C we obtain the same thermal ex-pansivity of the protein native state from spectroscopic and

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225101-6 Sassi et al. J. Chem. Phys. 139, 225101 (2013)

densitometric measurements. At higher temperatures, at thebeginning of the melting process, the lessening of M1 sug-gests that a rapid increase of protein volume could oc-cur and this is in contrast with density, photon correla-tion, and dielectric measurements indicating a shrink ofthe system given by the protein and its hydration shell.To reconcile this results one could assume that in the60–70 ◦C range the increase of effective protein volumeprobed by collective vibrations is compensated by the de-crease of hydration volume. Accordingly, one could re-fer to SF profile as a probe of effective molecular pro-tein volume, and to the apparent specific volume aslargely due to the hydration layer. This description findsa justification in the microscopic interpretation of volumedata.

The properties of the solvation layer enter the hydrody-namic and also the partial molar volume V◦ of a solute. Ac-cording to Chalikian40 this quantity can be divided into dif-ferent contributions assigned to the intrinsic (van der Waal)volume VM, the thermal volume VT, the interaction volumeVI and the isothermal compressibility βT of the pure solvent:

V◦ = VM + VT + VI + βTRT. (5)

The volume VT accounts for the imperfect packing of dif-ferent molecules (Vvoids) and the thermally activated fluctua-tions of structure induced by the solvent (Vvib). The effec-tive protein volume is the sum of VM and VT contributionsif one considers the space occupied by one solute moleculein a solvent cavity. Vvib can be roughly estimated by mul-tiplying the solvent-accessible surface area by the thickness(≈1 Å) of the empty space surrounding the protein.40 Thethermally induced denaturation determines an increase ofsolvent-exposed surface and then an increase of this term.This effect can compensate the cancellation of internal voids(decrease of Vvoids) occurring at melting, and can determinea larger effective protein volume. When this condition is ver-ified (VM + VT) increases with the unfolding of polypeptidechain: this is what we observe with the temperature behaviorof M1−1 at the beginning of melting range and further in thewhole high temperature domain.

The interaction volume, instead, takes into account theproperties of the solvation shell (βTRT is usually negligible).In particular, VI is a negative term representing the decrease ofwater volume due to interactions with exposed atomic groupsof the protein. In fact, X-ray and neutron scattering studies onpolypeptides indicate that the density of water around the pro-tein surface is roughly 10% larger than that of bulk water.32

Due to the increase of the solvent accessible surface, VI isfurther decreasing upon melting and this negative variationcan partially compensate the augment of (VM + VT). Accord-ing to this description the measured inflection of VLys at T= 60–70 ◦C might be attributed to this change of hydratingconditions, masking the increase of effective HEWL volumeprobed by Raman measurements.

IV. CONCLUSIONS

A 100 mg lysozyme/ml water solution has been studiedas a function of temperature in the 25–85 ◦C range where the

melting of secondary and tertiary structures occurs. A numberof striking results have been obtained from the comparison ofdensity and spectroscopic measurements.

For the first time the THz Raman profile of a proteinhas been analyzed as a function of temperature in the melt-ing range. The integrated intensity of this spectral feature hasresulted to be an efficient probe of the melting process sincea sharp increase is observed on increasing the content of theunfolded state of HEWL. This increase is reduced due to theoccurrence of a self-assembling process which is connectedto the strengthening of intermolecular contacts but not to thechange of molecular structure.

The arrangement of the molecular structure is probed bythe average frequency M1 of collective vibrations. This quan-tity shows a gradual change on increasing temperature in thepre-melting region, and a sharp drop of its value is measuredat temperatures where X-ray scattering experiment evidencesa drastic change of protein hydration shell.

The apparent specific volume of the native protein VLys ,as evaluated from density measurements, has a temperaturedependence in agreement with the one obtained for the hy-drodynamic radius by photon correlation spectroscopy:7 theyboth show a maximum at 50 ◦C.

A close relation exists between M1−1 and VLys for thenative state of HEWL: from these two quantities we obtainsimilar value of thermal expansion coefficient. Further stud-ies from this laboratory will extend this comparison to theunfolded and aggregated forms of the protein.

In accordance with the model proposed for interpretingthe low-lying vibrational modes of the native BSA, we sug-gest to consider HEWL molecule as a three dimensional,elastic nanoparticle with aperiodic charge distribution, anddescribe the SF χ ′′ profiles as a distribution of phonon-likevibrations. Following this description, we refer to the inverseproportionality of M1 respect to the volume of HEWL andobtain the thermal expansivity of the native state in perfectagreement with densitometric measurements. Moreover, werecognize a sharp increase of the protein volume at the be-ginning of the thermal unfolding, at temperatures where achange of hydrating condition determines a minimum of bothhydrodynamic radius and apparent specific volume. To rec-oncile these results we suggest M1 to be proportional to theinverse of effective protein volume, and VLys and Rh to belargely characterized by the properties of the solvation layer,at least in the 60–70 ◦C range. Thus, a straight connectionis established between microscopic and macroscopic proper-ties of the system with the possibility to describe the effectof heat denaturation on both protein structure and solvationlayer.

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