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Does a Dry Protein Undergo a Glass Transition? Anna V. Frontzek,*,†,‡ Serge V. Strokov,† Jan Peter Embs,‡ and Sergey G. Lushnikov†,§ †

A.F. Ioffe Physical Technical Institute, ul. Politekhnicheskaya 26, 194032 Saint-Petersburg, Russian Federation Laboratory for Neutron Scattering, Paul Scherrer Institut, CH-5232 Villigen, Switzerland § Saint-Petersburg State University, 199034 Saint-Petersburg, Russian Federation ‡

ABSTRACT: Bovine serum albumin (BSA) with extremely low hydration level 0.04, which is usually defined as dry, has been investigated in the temperature range between 200 and 340 K by incoherent inelastic neutron scattering using the neutron time-of-flight spectrometer FOCUS (PSI, Switzerland). Anomalous temperature behavior has been revealed for relaxational and low-frequency vibrational dynamics of BSA in the vicinity of 250 K. The mean-square atomic displacement has been shown to exhibit a change in the slope of temperature dependence near the same temperature. The presented results point out that the glass-like transition occurs in the dry protein.



INTRODUCTION

The other phenomenon exhibited by hydrated or solvated proteins is the dynamical transition.1 It is usually associated with the sharp rise in the mean-square atomic displacement around 200−240 K that is observable mostly by neutron scattering1,10,18−24 and Mössbauer spectroscopy.25 The mechanism of the dynamical transition has been a subject of hot discussion until now. There exist different points of view on the origin of the dynamic transition. It has been attributed to a change in the protein’s “effective elasticity”,26 to the microscopic manifestation of the glass transition in the hydration shell of proteins,6,27 to a fragile-to-strong crossover in the dynamics of hydration water,28 and to resolution effects due to a β-relaxation of the solvent (coupled to the protein) that enters the experimentally accessible frequency window.18,19 The interest in the nature of the dynamic transition has been stimulated by the fact that the measurable biological activity of proteins was found to appear around the same temperature.29 Recently, it was pointed out that the temperature of the dynamical transition Td of hydrated proteins and proteins in different viscose solvents is always higher than the glasstransition temperature Tg.16,17 Raman and inelastic neutron scattering spectra of both biopolymers10,30−34 and nearly all glass-forming systems in the glassy state17 exhibit a peak corresponding to acoustic-like excitations, the so-called “boson peak”. It is supposed that the boson peak appears due to disorder in glass-like systems. The nature of the boson peak still remains a topic of lively discussions in the scientific community. At high temperatures, the boson peak is hardly distinguishable from quasi-elastic

The dynamics of proteins and other biopolymers plays an important role in their biological function. The intrinsic flexibility of proteins provides the possibility to adjust the conformational state to the one that is favorable for interaction with substrates, ligands, and other molecules. The relation between dynamics and function mechanisms initiated the intensive studies of macromolecule dynamics undertaken in the past decades. It has been found that there exist a lot of similarities between the dynamics of proteins and glass-forming systems.1−3 The glass transition in hydrated proteins can be observed by calorimetry,4−7 measurements of thermal expansion,8 and other techniques.9−12 For example, the pronounced step-like temperature behavior of heat capacity that is a typical feature of glass transitions has been demonstrated for lysozyme crystals with different levels of water content.5 The rise in hydration level of protein powders leads to a decrease in glass-transition temperature Tg because of the plasticizing effect of water.5 Dry lyophilized protein powders exhibit strongly suppressed dynamics and do not manifest a biological activity.13,14 However, it still remains questionable if the glass transition can occur in dry biopolymers. The dynamics of hydrated biopolymers near Tg is supposed to exhibit an activation of additional anharmonic motions and, as a consequence, a change in the temperature dependence of the mean-square atomic displacement . The mean-square displacement weakly changes with temperature below Tg and shows stronger temperature dependence above Tg. Such a change in the (T) dependence is a common feature in the dynamics of biopolymers15,16 and conventional glass-forming systems.17 © 2014 American Chemical Society

Received: October 23, 2013 Revised: February 12, 2014 Published: February 21, 2014 2796

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deuterium, which exhibits comparable values of coherent (5.6 barn) and incoherent (2.0 barn) scattering cross sections, there is no need for future separation of both contributions preliminary to the data analysis. To examine if the glass or the dynamical transition occurs in dry BSA powder, we have calculated at different temperatures (the results are presented in Figure 1) using the

contributions that appear as a result of relaxational processes activated with increasing temperature. In conventional glassformers, the maximum of the boson peak shifts towards zero energy transfer E = 0 for increasing temperatures.17,35 Recently, similar temperature behavior of the boson peak has been shown for hydrated lysozyme.10 In previous studies, the glass transition as well as the dynamical transition could not be clearly observed in the case of lyophilized protein. Therefore, it was important for us to understand if glass-like or dynamical transitions really could occur in a dry protein. We applied incoherent inelastic neutron scattering to investigate the dynamics of a dry bovine serum albumin (BSA) that was used as model system in our experiments. The applied experimental technique allows us to identify even tiny changes in fast protein dynamics. The relaxational and low-frequency vibrational processes of BSA have been studied in the temperature range where the glass and the dynamical transition normally occur.



MATERIAL AND METHODS Sample Preparation. The BSA has been purchased from Sigma-Aldrich and used for neutron scattering experiments without further purification, lyophilization, and deuteration of the protein. The BSA powder was placed in a cylindrical aluminum can; an indium wire was used to seal the can. The protein hydration level (gram of water per gram of protein) has been determined by thermogravimetric analysis to be of 0.04, which is much lower than the water content needed to form the primary hydration shell of protein molecules. The protein powders with such a hydration level are usually defined as a dry protein.13,18 Since it is impossible to remove the water completely without disruption of the native spatial protein molecular structure,13,36,37 the BSA powder under study has included water composed of a few water molecules that are tightly bound to the charged side chains on the surface of protein molecules and a fraction of internal water confined within small cavities of a protein structure.13 Neutron Scattering Experiment. The neutron scattering experiments have been performed at the Paul Scherrer Institute (Switzerland) using the cold neutron time-of-flight spectrometer FOCUS at the Swiss spallation source SINQ. The energy resolution (fwhm) of the spectrometer was 100 μeV (incident wavelength λi = 4.3 Å). First, the temperature of the sample was decreased to 30 K. Then, the scattered neutrons were collected during 3 to 4 h for each spectrum in the temperature range between 200 and 340 K. All protein spectra were normalized to vanadium and corrected for the scattering from the empty can. The obtained results have been analyzed using the program package DAVE.38 Neutron Scattering Data Analysis. Estimation of MeanSquare Atomic Displacement. We have studied the protein powder with an extremely low hydration level; therefore, the neutrons were predominantly scattered incoherently from hydrogen atoms of protein molecules. The amount of hydrogen atoms belonging to water molecules relative to all hydrogen atoms in the system was estimated to be ∼6.2%, which is negligibly small and cannot provide a relevant contribution into the scattering process. The hydrogen-to-deuterium exchange in the protein has not been performed due to our interest in uncorrelated fast dynamics of the protein, which can be examined by the analysis of incoherent contribution. Since the incoherent scattering cross section of hydrogen (80.3 barn) is much bigger than the coherent (1.8 barn), in contrast with

Figure 1. Temperature dependence of the mean-square atomic displacement of bovine serum albumin with hydration level 0.04. The inset shows an example how the mean-square atomic displacement is derived from the linear fit of the logarithmic integral intensity of the incoherent elastic scattering (normalized to the integral intensity of elastic peak obtained at 30 K) as a function of Q2.

standard procedure that implies analyzing the elastic contribution in the dynamic structure factor S(Q,ω). The experimentally determined structure factor is given as S(Q , ω) = Stheo(Q , ω) ⊗ Sres(Q , ω)

(1)

where Sres(Q,ω) is the instrumental resolution function and Stheo(Q,ω) is the theoretical dynamic structure factor applied for data fitting as a model function. If different relaxational motions are uncorrelated, then the following equation can be used to represent the theoretical incoherent dynamic structure factor: 2

2

Stheo(Q , ω) = e−⟨x ⟩q [A 0(Q ) ·δ(ω) n

+

∑ Ai(Q )·Li(Q , ω)] i=1

(2)

where the A0(Q) is an elastic incoherent structure factor (EISF), Ai(Q) is a quasi-elastic incoherent structure factor (QISF) of some certain quasi-elastic component, δ(ω)is a delta-function, and Li(Q,ω) is a Lorentzian. The elastic line contains the contribution of all motions that are slower than the characteristic time τspectrometer of the spectrometer. If the dynamic structure factor S(Q,ω)is analyzed in broad energytransfer range, then two quasi-elastic components can clearly be distinguished for dry BSA. (See Figure 2.) However, the meansquare atomic displacement calculations require a precise estimation of the elastic scattering only. Therefore, the energytransfer range used to determine was set to −0.5 < ω < 0.8 meV. In this case, the broad quasi-elastic component can simply be assigned to a linear background. Finally, only one 2797

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account the neutron scattering in vanadium. An example of incoherent dynamic structure factor of BSA S(ω) measured at 240 K is depicted in Figure 2. It is seen that the elastic contribution, two quasi-elastic components, and the boson peak can be clearly distinguished in S(ω). The information regarding the contributions of all dynamical processes and their temperature evolution has been obtained from S(ω) analyzed at different temperatures. The elastic line has been fitted by a Gaussian because this function is the result of a delta-function convoluted with the resolution function of FOCUS. In addition to the elastic scattering, two relaxational components, which contribute to the quasi-elastic scattering (see Figure 2), can be distinguished. Both quasi-elastic components have been fitted by Lorentzians. However, it is necessary to keep in mind that protein dynamics covers a broad distribution of time-scales. Therefore, the two relaxation components give an impression about the average dynamics of slow and fast protein relaxations that can be, in fact, a superposition of motions with different characteristic relaxational times18,40 or a stretched complex relaxation process, as it has been shown previously for the main relaxational process in hydrated lysozyme.18 The fast relaxational component has broader full width at half-maximum (fwhm) and can be associated with motions of some aminoacid residues. The slow relaxational component is more narrow than the fast one and probably originates from the relaxations involving bigger parts of protein molecule, for example, several amino-acid residues, some part of polypeptide chain, and elements of secondary structure or even protein domains. The nature of both quasi-elastic components will be discussed in the next section. A relaxational time is inversely proportional to the width of a corresponding quasi-elastic component and can by calculated by the equation:

Figure 2. Incoherent dynamic structure factor of dry BSA measured at 240 K and its fitting. The red line shows the final curve obtained as a result of data fitting, the green line is the fitting of elastic line by Gaussian, the orange line corresponds to the log-normal distribution used for approximation of the boson peak, and the blue and violet lines are the Lorentzians describing the narrow and broad quasi-elastic components, correspondingly.

Lorentzian was used for fitting of quasi-elastic scattering. The following equation was used for the dynamic structure factor Stheo(Q,ω) analysis: 2

2

Stheo(Q , ω) = e−⟨x ⟩Q [A 0(Q ) ·δ(ω) + A1(Q ) ·L1(q , ω)] + B

(3)

where A1(Q)and L1(Q,ω) correspond to the narrow quasielastic component and B is the background. The mean-square atomic displacement has been directly derived by the approximation of elastically scattered neutrons as a δ-function and analyzing its Q dependence in the range from 0.4 to 1.8 Å−1: Stheo(Q , ω ≈ 0) ∼ exp( −⟨x 2⟩Q 2)

τ=

2π ℏ fwhm

(5)

According to the fluctuation−dissipation theorem, the spectral function S(ω) is proportional to the imaginary part of susceptibility χ″(ω) S(ω) ≈

(4)

The procedure for the data analysis using eq 4 is shown in the inset in Figure 1: the value of can directly be calculated from the slope of ln[Stheo (Q,ω ≈ 0)] presented as function of Q2. All Stheo (Q,ω ≈ 0,T) values for different temperatures were normalized to Stheo (Q,ω ≈ 0, T = 30 K) measured at 30 K. The temperature dependence of obtained according to the procedure above described is shown in Figure 1. Analysis of Incoherent Dynamic Structure Factor. The incoherent dynamic structure factor S(Q,ω) of BSA has been obtained at different temperatures in order to study the temperature evolution of low-frequency vibrational and relaxational processes. S(Q,ω) was analyzed at energy transfers up to 20 meV. According to estimates made in ref 39, the multiphonon scattering contribution is insignificant at low frequencies. Moreover, it has been previously demonstrated for proteins that the shape of S(Q,ω) does not depend on wave vector Q.14 Therefore, the neutron scattering data for different scattering angles can be corrected for self-shielding and the contribution of the aluminum can, and then be simply summarized for all Q values and normalized taking into

⎛ T ∝⎜ ⎝ I

kBT χ ″(ω) for ℏω ≪ kBT ℏω

∫0



⎞−1 χ ″(ω) dω⎟ ∝ χ ′(0)−1 ⎠ ω

(6)

(7)

where kB is the Boltzmann constant and T is the temperature. So, the integral intensity of quasi-elastic scattering I in neutron spectra is proportional to the static real susceptibility χ′(0) at the lowest frequency limit of neutron scattering spectra. The integral intensity of a quasi-elastic component is a nonnormalized quasi-elastic structure factor Ai(Q) according to the definition of Ai(Q) (see above). The boson peak manifests itself in S(ω) as a vibrational bump present in the discussed frequency range. It is especially pronounced at low temperatures where the contribution of quasi-elastic scattering is less dominant (Figure 3). It was demonstrated that the shape of the boson peak of globular proteins can be described by a log-normal distribution because it appears as a result of a distribution of quasi-localization lengths for acoustic waves within the protein.31 Hence, we used a log-normal distribution ln N(ω) to fit the boson peak: 2798

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Figure 3. Dynamic structure factors of dry BSA measured at different temperatures. 2⎤ ⎡ 1 ⎛ ln ω/ωBP ⎞ ⎥ S(ω) ≈ ABP exp⎢ − ⎜ ⎟ ⎢⎣ 2 ⎝ δωBP ⎠ ⎥⎦

Figure 4. Temperature dependence of the frequency of boson peak maximum ωBP for dry BSA powder.

(8)

where ABP is the amplitude, ωBP is the frequency that corresponds to the peak maximum, and δωBP is the parameter characterizing the width of the log-normal distribution. As a result, the final equation used to fit S(ω) in the energytransfer range from 0 to 20 meV was a sum of the following functions: (i) a Gaussian G(ω) used to fit the elastic peak, (ii) two Lorentzians that correspond to the slow Lslow(ω) and the fast Lfast(ω) relaxational components, (iii) a log-normal distribution ln N(ω) to fit the boson peak, and (iv) the background B S(ω) = G(ω) + A s ·Lslow (ω) + A f ·Lfast(ω) + ln N (ω) + B

(9)

The fitting procedure has been performed in such a way that if the correlation coefficient R had value less than 0.95 then the final fitting parameters were rejected as inappropriate. Such an approach for data analysis gave us the possibility to investigate the temperature evolution of the boson peak maximum (Figure 4), the characteristic times of the both relaxational components, and their integral intensities or, in other words, susceptibilities (Figures 5 and 6).

Figure 5. Arrhenius plot for the characteristic time of slow relaxational process found in dry BSA (red squares) and the integral intensity of quasi-elastic component corresponding to this process (blue circles). The dashed red and blue lines are the guidelines for eyes.



RESULTS AND DISCUSSION The temperature dependence of the mean-square atomic displacement for BSA powder with a hydration level of 0.04 is shown in Figure 1. The value of the obtained is in good agreement with previous results obtained for dry lysozyme powder by Tsai et al.41 and Sokolov et al.18,42 In numerous studies, it was pointed out that of dry proteins measured by neutron spectroscopy increases monotonically with temperature over a broad range up to 300−350 K.15−17,41 Contrary to this statement, it can be clearly seen in Figure 1 that there exists a slight change in the slope of for dry BSA: it becomes steeper for increasing temperature after crossing 240−260 K. It is probably hardly observable if the data are presented on different scales that cover broader temperature intervals and values. The anomaly in (T) dependence of dry BSA can be associated either with the glass or the dynamical transitions.15−17 The existence of the dynamical transition in dry proteins is doubtful. Despite the fact

Figure 6. Arrhenius plot for the characteristic time of fast relaxational process observed for dry BSA (red squares) and the integral intensities of quasi-elastic scattering corresponding to this process (blue circles). The dashed red and blue lines are the guidelines for eyes.

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relaxation time of the main relaxation in hydrated lysozyme observed by dielectric and neutron spectroscopy.23 The nature of this process, however, remains unknown. It is supposed that it involves a significant part of protein molecules but is characterized by limited atomic displacement of ∼3 Å23 One interpretation relates this relaxation to the secondary structure of protein molecules.45 It is difficult to directly draw an analogy between the slow relaxation process of dry BSA found in our study and the main relaxation of hydrated lysozyme reported by Sokolov et al.18,23 because the main relaxation in hydrated proteins has been established to depend strongly on hydration level: the main relaxation of dry lysozyme was found to be slower by five to six orders of magnitude than for hydrated lysozyme.18 Therefore, the slow relaxation process in dry BSA and the main relaxation found by Sokolov et al.18,23 in a hydrated protein are most probably different in origin. Nevertheless, we would also associate the slow relaxation process in dry BSA with the relaxation of some protein molecule parts or secondary structures. The fast relaxation process of dry BSA is also characterized by an anomalous temperature dependence of the relaxation time τfast (Figure 6). In the low-temperature range up to 240 K, the relaxation time of the fast process decreases with increasing temperature, then its value drops and starts to increase above 260 K. The absolute value of τfast is comparable to the relaxation time of methyl groups estimated for some other proteins.23 The rotations of methyl groups buried in the core of protein molecule become activated at very low temperature and are not sensitive to hydration. So we can suggest that the fast relaxation process in the dry protein found in our study originates from the methyl group relaxational motions. A disordered molecular configuration of the liquid becomes frozen-in below Tg: molecules vibrate near the center of mass and do not undergo translational diffusion. The definition of a glass transition implies that time scales of a structural αrelaxation reach 1−100 s in the vicinity of Tg.17,46 Both relaxation processes revealed in dry BSA in our study have much shorter characteristic times while the system is approaching a glass transition; therefore, none of them can be considered to be α-relaxation process. The pronounced slowing down of slow relaxational process below 250 K (Figure 5) reflects the reduction in molecular mobility of some structural elements of a protein molecule that is unusual for a glass transition. However, the singularity in τslow (T) dependence can be also associated with the activation of different relaxation process than the one observed in low-temperature region.47 In complex glass-forming systems like glycerol−water systems with concentrations of glycerol below 40%, similar anomalies have indicated the additional relaxation processes due to ice particles and interfacial water.47 Since the BSA powder contained a quite limited amount of water molecules, the anomalous activation of relaxational motions in lowtemperature might be caused by intrinsic properties of complex protein structure. It might occur that sizes of protein molecules involved in relaxational fluctuations reduce with decreasing temperature or the value of activation energy needed for “barriers crossing” between different conformational states changes. In contrast with the slow relaxation process, the characteristic relaxation time of methyl groups τfast significantly reduces with increasing temperature between 240 and 260 K (Figure 6), which is most likely a result of a glass-like transition in the protein. However, the reason for the following increase in τfast value above 260 K is unclear. Similarly to the slow

that there are different points of view on its origin, it has been demonstrated that the dynamical transition is strongly influenced by protein hydration. The glass transition has been identified for protein solutions,7,11,12 for hydrated protein powders,10−12 and in protein crystals dehydrated down to 7.4% of water content.5 However, there was no evidence found that a glass transition can occur in a dry protein. To answer the question of the reason for the anomalous behavior in the vicinity of 250 K, we have investigated the low-frequency vibrational and relaxational dynamics of dry BSA in detail. Considering the dynamic structure factor S(ω) of dry BSA at different temperatures (Figure 3), it can be noticed that the contributions of the boson peak and the quasi-elastic scattering are clearly distinguishable in all neutron spectra up to 300 K. This is not the case for conventional glass-formers, which usually exhibit the boson peak at temperatures below Tg: at temperatures near and above Tg, the increasing quasi-elastic contribution tends to obscure the boson peak.17 The fact that the boson peak is observed for spectra of proteins even at high temperatures can be caused by the intrinsic molecular structure of a protein.43 Despite conformational rearrangements that are important for carrying out the specific biological function, the average compact 3-D structure is maintained below the thermal denaturation temperature for the majority of proteins. This creates favorable conditions for quasi-localization of acoustic waves on the length scales of a protein molecule.31 The temperature dependence of the boson peak maximum of dry BSA is shown in Figure 4. It is clearly seen that the boson peak shifts towards lower energies with increasing temperature. A similar tendency of the boson peak temperature behavior has been demonstrated for hydrated lysozyme in Raman spectroscopic investigations.10 However, in the case of dry BSA, a sharp step-like anomaly in the temperature dependence of the frequency of the boson peak maximum near 240−260 K can be observed. Moreover, an apparent slope of the curve dωBP/dT becomes significantly steeper above ∼250 K. A similar variation in a boson peak position has been revealed for glass-forming systems near Tg.35 Therefore, it can be concluded that dry BSA undergoes a glass-like transition in the vicinity of 250 K, and an anomalous behavior in d/dT at the same temperature previously discussed is also a feature of this transition. Because the boson peak is assumed to provide information regarding the elasticity on length scales of a few nanometers43,44 that is comparable to sizes of BSA molecules, the observed singularity in the temperature behavior of the boson peak found for dry BSA is most likely associated with the remarkable change in rigidity/elasticity of a whole protein molecule near 250 K. The quasi-elastic scattering observed in dynamic structure factor S(ω) of dry BSA is associated with relaxational processes in the system. We can identify two relaxational components in the measured neutron spectra: a narrow component that corresponds to a slow relaxation and a broad component that corresponds to a fast relaxation. The values of the relaxation times for both components are presented as Arrhenius plots in Figures 5 and 6. It is obvious that a representation of relaxation time data on logarithmic scales does not provide any additional information in comparison with the linear scale because of limited intervals of times. The relaxational time of the slow relaxation τslow decreases monotonically with temperature up to 240 K, followed by a discontinuity (Figure 5). After a sharp increase, τslow continues to decrease with temperature again monotonically in the range between 260 and 340 K. The absolute value of τslow of dry BSA is comparable to the 2800

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recently by Capaccioli et al.15 and Ngai et al.16 based on the socalled caged dynamics. This feature of protein dynamics seems to be similar to conventional glass-forming systems where the caged molecular motions and the sensitivity of amplitudes of these motions to the change in the specific volume while crossing Tg are supposed to be responsible for anomalies in d/dT. At the same time, the authors do not exclude other possible origins of the observed change of the slope in (T) such as vibrational contributions.15 Indeed, the boson peak frequency of dry BSA powder is shown here to be sensitive to crossing Tg (particularly, the maximum of the boson peak shifts toward the elastic line at temperature increase) that can affect the intensity of elastic peak. In summary, we have revealed the nontrivial temperature behavior of the mean-square atomic displacement and vibrational and relaxational dynamics of lyophilized BSA powder: the mean-square displacement exhibits the change in the slope in the vicinity of 240−260 K; the maximum of the boson peak shifts drastically towards zero energy transfer at the same temperature; and finally, the relaxation times of the slow and the fast relaxation processes as well as the susceptibility of the slow relaxation undergo anomalous behavior also near 250 K. All of these changes revealed in protein dynamics indicate that a glass-like transition occurs in the dry protein.

relaxation process, two possible scenarios could be supposed. One of them is a change in the volume available for methyl group relaxation and, therefore, amplitudes of motions with increasing temperature because of activated fluctuations of neighboring amino acid residues. The other explanation is an alteration in energy barriers of methyl group rotations. As a result, the methyl group motions are slowing down. In any case, we expect that an increase in τfast above 260 K occurs only in a limited temperature interval. The activation of internal molecular mobility of dry BSA as a function of temperature was investigated by determination of the integral intensities of the quasi-elastic components. As it was shown in Material and Methods, the integral intensity is proportional to the real part of the susceptibility (according to eqs 6 and 7). We can also identify the integral intensity of quasi-elastic scattering as a non-normalized quasi-elastic structure factor. (See eq 2.) The temperature dependences of integral intensities for the fast and slow relaxations in dry BSA are shown in Figures 5 and 6 as blue signs. The main feature of the both temperature dependences is a rising contribution of relaxational motions with temperature. The integral intensity of the broad quasi-elastic component (fast relaxation process) changes monotonically in the whole investigated temperature range and does not exhibit any anomalies (Figure 6). So, the activation of methyl group relaxation occurs gradually and is not affected by the glass-like transition in the protein in the vicinity of 250 K. In contrast with the fast relaxation, the slow process is characterized by a sudden increase in the susceptibility near 250 K (Figure 5). This conclusion can be made based on the change in the slope of the integral intensity temperature dependence of the narrow quasi-elastic component. Similar temperature behavior has been previously revealed for quasi-elastic structure factors at the dynamic transition in hydrated purple membranes.24 The authors have identified three quasi-elastic components. The quasi-elastic structure factors of all of them start to increase drastically at different temperatures in the range from 170 to 260 K. However, the authors mainly discussed the critical role of water in its contribution in the dynamic transition. We have investigated the dry protein powder. So, we emphasize that a sharp increase in the contribution of slow relaxation process near 250 K is caused by changes in protein dynamics associated only with a glass-like transition. We exclude the dynamical transition as a possible explanation for the anomalous relaxational and low-frequency vibrational dynamics of dry BSA. First of all, the dynamic transition temperature identified as a change in the slope d/dT has been shown to be dependent on the resolution of the spectrometer, 18,19 which is not the case for a glass transition.15,16 According to our results, all dynamical processes observed in different frequency ranges undergo the anomalous behavior at the same temperature; therefore, it is doubtful that the resolution of the spectrometer could affect the transition temperature. The other contradicting point is the critical influence of water on the dynamical transition. Despite the fact that there exist different points of view on the origin of the dynamical transition, mostly all of them assign the main role to the water in hydration shell of protein.6,18,19,27,28 Since the water content of the BSA powder studied here was extremely low, we tend to attribute the critical dynamics near 250 K to the glass-like transition in protein itself. The existence of a kink in d/dT of solvated and hydrated proteins at a glass transition has been explained



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]ffe.ru. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work is based on experiments performed at the Swiss spallation neutron source SINQ, Paul Scherrer Institute, Villigen, Switzerland. We thank Ekaterina Pomjakushina for the performance of thermogravimetrical analysis for BSA powder. The study was supported by Russian Foundation for Basic Research, grants No. 10-02-00511-a and 11-02-00016-a.



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