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Soft Matter
COMMUNICATION
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aISIS Neutron and Muon Facility, Ruthe
Technology Facilities Council, Didcot,
[email protected] Center for Neutron Research, Nationa
Gaithersburg, MD 20899, USA. E-mail: josepcPolymers Division, National Institute of Stan
20899, USA. E-mail: [email protected] Sciences Division, Oak Ridge Na
USAeDept. of Chemistry, University of Tennes
[email protected] Science, University of Akron, Akron
† Electronic supplementary informa10.1039/c3sm50492a
‡ Current address: Faculty of Applied ScDel, The Netherlands. s.khodadadi@tud
§ Current address: National Research CResearch Laboratory, Code 6120, Chemistr
Cite this: Soft Matter, 2013, 9, 5336
Received 17th February 2013Accepted 8th April 2013
DOI: 10.1039/c3sm50492a
www.rsc.org/softmatter
5336 | Soft Matter, 2013, 9, 5336–53
Solvent effects on protein fast dynamics: implicationsfor biopreservation†
Victoria Garcıa Sakai,*a Sheila Khodadadi,‡b Marcus T. Cicerone,c Joseph E. Curtis,b
Alexei P. Sokolovde and Joon Ho Roh§*f
In the context of biopreservation, we study the influence of water,
glycerol and trehalose on the ps–ns dynamics of lyzosyme using
neutron scattering. Results indicate that the choice of bioprotectant
depends on the storage temperature; glycerol is the most effective
for low temperatures and trehalose for high temperatures.
Proteins undergo constant structural uctuations, the ener-getics of which are inuenced by the surrounding solvent. Thenative solvent – water – is essential for life as we know it.However, some plants and organisms have adapted to survive inits absence or at temperatures well below the freezing point.1,2
Such systems accumulate high concentrations of viscous sugars(e.g. trehalose) or polyalcohols. Although trehalose and otherdisaccharides are routinely used in the biopharmaceuticalindustry, the mechanism for stabilization and its relationshipwith water remain unclear. A thorough understanding of theinuence of solvent on protein dynamics at a molecular level isthus essential both for elucidating the mechanisms of proteinfunction and for nding pathways to control protein stability
rford Appleton Laboratory, Science &
OX11 0QX, UK. E-mail: Victoria.
l Institute of Standards and Technology,
dards and Technology, Gaithersburg, MD
tional Laboratory, Oak Ridge, TN 37831,
see, Knoxville, TN 37996, USA. E-mail:
, OH 44325, USA. E-mail: joonho.roh.ctr.
tion (ESI) available. See DOI:
iences, Del University of Technology,el.nl
ouncil-Research Associate, US Navaly Division, Washington DC 20375, USA.
40
and activity. In this contribution we study the effect of threesolvents, water, glycerol and trehalose, on the dynamics of themodel protein lyzosyme. The data show a clear demarcation ofthermostability with temperature and point to an appropriatesolvent choice: glycerol at low temperatures and trehalose athigh temperatures.
The simplest evidence of the role of a solvent on proteindynamics is the dynamical transition, TD,3,4 dened as the onsettemperature for anharmonic motions on the ns-time scale andobservable in neutron scattering (NS) experiments as a sharprise in mean square displacement (MSD). While absent in dryproteins and present at certain hydration levels, its existence asa ‘transition’ has been the subject of great debate,4–6 as well asits relevance to protein activity, with some reports suggestingthat it coincides with the onset of measurable protein activity,7
despite observation of a similar rise in MSDs for unfoldedproteins and RNA.8 What is clear is the idea that protein andsolvent dynamics are strongly coupled: activation of solventtranslational motions at temperatures above their criticaltemperature is the key for triggering the TD.9–13
Proteins embedded in water or glycerol show TDs at around200 K and 270 K respectively,14 where the solvents begin tobehave like liquids above their critical temperature. In contrast,trehalose-embedded proteins do not show the dynamic transi-tion up to room temperature (RT) owing to its high glass tran-sition temperature (Tg) of 390 K. This is believed to be the mainreason for the superiority of trehalose for preservation: highviscosity at RT results in the immobilization of the biologicalstructure and its insensitivity to moisture.15 However, it is bothTg and fragility that determine the efficiency of a glass for bio-preservation.12,16 Fragility is a traditional characteristic of thenon-Arrhenius temperature dependence of structural relaxationsa(T) in glass-forming liquids.17 Systems with nearly Arrheniustemperature dependence of sa(T) are called strong, while theones with strongly non-Arrhenius behaviour are called fragile.The addition of glycerol, a strong glass former, leads to largerenergy barriers for conformational transitions in the proteinthan in the fragile glassy trehalose.12 Furthermore, the addition
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Fig. 1 MSDs of dry lysozyme (DL), hydrated lysozyme (WL), lysozyme in glycerol(LG), and lysozyme in trehalose (LT) corrected for trehalose contribution (see ESI†),up to 300 K. The inset presents the data for DL and LT extended up to 370 K; PTand LT0 are the pure trehalose and uncorrected trehalose–lysozyme data.
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of glycerol to a trehalose matrix, despite lowering the Tg of thesystem, has proved to prolong the shelf-life of proteins andenzymes.16,18 Moreover, at lower temperatures glycerol slowsdown protein motions more than trehalose. An example is theinuence of glycerol and trehalose on the kinetics of CO-myoglobin geminate rebinding: at T < 270 K the escape rate ofunbound CO is faster in proteins placed in solid trehalose thanin liquid glycerol.19
Solvated proteins present a diverse relaxation map, withtimescales ranging from milli- to femtoseconds, and assign-ment of each process is non-trivial. Our previous studies frommolecular dynamics simulations,20–22 NS23 and dielectric spec-troscopy24 (DS) revealed ve main dynamical processes: low-frequency collective vibrations (the Boson peak), fast confor-mational uctuations, methyl group rotations, a slow processinvolving both the protein and hydration water and a slowerglobal motion involving the entire protein structure. The slowrelaxation process, where solvent–protein coupling is relativelystrong, is responsible for TD and possibly related to the activa-tion of biological functions.25 The precise effect of solvents onthe various dynamical processes remains unclear, except formethyl group dynamics which are independent of hydra-tion.21,23,24 In this contribution we address the effects ofthree important solvents, namely water, glycerol and trehalose(with viscosities at 20 �C: trehalose >1015, glycerol 1200, water 1[mPa s]), on the dynamics of the model protein lysozyme and ingeneral on the stabilization of biological molecules.
Lyophilized hen-egg white lysozyme was used aer beingdialyzed to remove salts. The lyophilised powder was washed inD2O to deuterate all exchangeable H-atoms and then re-lyoph-ilized. Deuterated glycerol, D2O, and partially deuteratedtrehalose (user-made at 63 � 3% deuteration level) were used toreduce the neutron scattering signal from the solvent, andprobe primarily the dynamic response from the protein. Theweight ratio of D2O, glycerol and trehalose to the weight oflysozyme was 0.5 : 1, 1 : 1 and 1 : 1, respectively. NS spectrawere collected at 50 < T (K) < 370 K on two spectrometers, HFBSand DCS, at the NIST Center for Neutron Research (see ESI†),covering a combined time range between �0.05 ps and 2 ns.Elastic scattering temperature scans were also performed onHFBS at a rate of 0.7 K min�1 to estimate the mean-squareddisplacement, hr2(T)i. Due to a much higher incoherent scat-tering cross-section of H-atoms which are well-distributedwithin the protein, the measured neutron scattering spectramostly reect internal motions in the protein according to theself-correlation principle. We note that in the lysozyme–treha-lose mixture, owing to the partial deuteration of the trehalose,one third of the scattering signal arises from the non-exchangeable hydrogen atoms and so it needs to be corrected(see ESI† for further experimental, data correction and analysisdetails). Data were corrected for detector efficiency, empty cellscattering and normalised to protein mass.
Fig. 1 shows the MSDs determined using the Gaussianapproximation. All protein samples show a change in slope at�100 K (more clearly visible in the inset and in the ESI, Fig. S1†)that is independent of solvent. The onset at T � 100 K has beendiscussed in many papers and is ascribed to methyl group
This journal is ª The Royal Society of Chemistry 2013
rotations.21–24 Only hydrated lysozyme (WL) and lysozyme inglycerol (LG) samples show a dynamical transition at TD of�200K and 275 K, respectively. TheMSD for the lysozyme in trehalose(LT) sample follows the dry protein (DL) up to �270 K, abovewhich hr2i in LT is lower, suggesting that protein dynamics aremore suppressed than in the dry state.
Fig. 2 (top panel) shows the low-frequency dynamic struc-ture factor S(Q,n) measured on HFBS at 250 K for wet and drylysozyme, and lysozyme in glycerol. Our analysis shows that thewidth of the quasielastic spectra is essentially Q-independent,while the intensity increases strongly with Q. This is in agree-ment with earlier studies21–24 and indicates the localized natureof the motion. DL shows some quasi-elastic broadening asso-ciated with CH3 rotations,23 which still dominates and is seenunaffected in LG. In contrast, water allows more exibility inthe protein as observed by the larger broadening. Increasingthe temperature to RT (middle panel) leads to an increase inbroadening of DL and LT as expected for CH3 rotations. LGshows even larger broadening because the temperature of 320K is 50 K above the TD of the LG system. The hydrated proteinexhibits the strongest signal being at �95 K above its TD.Finally, increasing the temperature to 370 K (bottom panel)shows quasi-elastic scattering for DL smaller than at 295 Kowing to the CH3 rotations moving out of the HFBS window.We note that the difference of MSDs between LT (or DL) andpure trehalose (PT) (inset Fig. 1) is due to the methyl groupdynamics that are absent in trehalose. More important,however, is that the quasi-elastic intensity in LT is smaller thanthat in DL, which is consistent with the MSD data (inset ofFig. 1). The excess mobility in DL at 370 K must arise fromother relaxation processes approaching the nanosecond timewindow at this temperature. Considering that the melting ofdry lysozyme (�4.8 wt% water) takes place at �390 K,26 thesmall intensity at the accessed time scale possibly reects theonset of the denaturing process. Trehalose suppresses thesemotions and provides stabilization of the native structure oflysozyme at such high T.
Soft Matter, 2013, 9, 5336–5340 | 5337
Fig. 2 Neutron scattering spectra of DL, WL, LG, LT, and PT at various temper-atures summed over allQ (data fromHFBS). Instrumental resolution is shown witha vanadium standard.
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The high-frequency (10 GHz to 3 THz) S(Q,n) spectrameasured using DCS are presented in Fig. 3. At T ¼ 150 K weexpect a negligible contribution from CH3 rotations at these
Fig. 3 Neutron scattering spectra of DL, WL, LG, and LT at various temperatures.Spectra are summed over all Q (DCS data).
5338 | Soft Matter, 2013, 9, 5336–5340
frequencies, and the quasi-elastic signal is dominated bythe fast conformational uctuations at n < 300 GHz, and thevibrational Boson peak at n� 1 THz. The spectra of DL and LT atT ¼ 150 K have the highest quasi-elastic intensity. In contrast,LG shows strongly suppressed dynamics consistent with theprevious results from NS11 and Raman scattering.14,23 Atambient temperature, WL shows the strongest quasi-elasticintensity (bottom panel). At 320 K LG exhibits a slightly higherintensity than LT. The latter shows an even weaker intensitythan DL at 295 K, despite the temperature difference. Theseresults emphasize the advantage of trehalose in suppressingprotein dynamics at high T.
Based on these results we propose the following picture. At T< 200 K high-frequency dynamics are dominated by picosecondconformational uctuations and these are strongly suppressedin glycerol, slightly in water and not at all in LT, compared withdry lysozyme. Small glycerol molecules form rather rigid well-packed structures with very weak uctuations. They are able tointeract very efficiently with the protein surface.27 This createsreduced cage sizes and protein motions are strongly sup-pressed. In this case, it is the property of a strong glass former(suppressed fast conformational uctuations28) that is respon-sible for effective low-temperature protection provided by glyc-erol. Such an effect has also been observed in simulations forthe surface residues.10 It is known that the structure of proteinsin glycerol is similar to that in water.29,30 A reduction in the cagesize could also explain the slight suppression of fast modes seenin water as compared to the dry protein (Fig. 3, top). Thestronger suppression by glycerol than by water can be explainedby its higher bulk viscosity. Simulation studies11 showed thatprotein dynamics is strongly coupled to glycerol motions.
Trehalose on the other hand shows a slight soening of thefast modes at low temperatures. A similar result was obtained byRaman scattering.31 At 150 K trehalose is deep in the glassystate, even more so than glycerol or water, but trehalose mole-cules are much bulkier and larger in size, packed less efficiently.As a result fast conformational uctuations in pure trehaloseremain strong even in a glassy state12 (common for fragile glassformers28). Due to their bulkiness, trehalose molecules alsocannot interact well with the protein surface. This means largercage sizes providing an extra volume for the protein atoms torattle about.
This picture changes at higher temperatures and mightexplain the use of trehalose as a stabilizer at RT (or higher). Fastconformational modes of protein in LT are more suppressed ascompared with DL, WL and LG (Fig. 3, bottom panel). Atambient temperatures, water and glycerol are well above theirTgs and facilitate the protein dynamics. In this case, the solventviscosity controls the dynamical behavior and also explains themuch stronger dynamics in water than in glycerol.
At even higher temperatures, where the protein is expected todenature, the efficacy of trehalose is the greatest. Owing to itshigh Tg of 390 K, fast picosecond dynamics are suppressed andthe protein remains protected from unfolding.
We nally comment on the whole feature of slow proteindynamics. Consistent with experimental suggestions,25 recentsimulations of lysozyme32 have shown threemain contributions:
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(i) methyl rotations, (ii) localized diffusion and (iii) jumps. Thelatter are outside the frequency range accessible to HFBS. Attemperatures below the dynamical transition, CH3 motionsdominate the nanosecond time-regime and are unaffected by thesolvent. Hydration of proteins facilitates an additional relaxa-tion, which involves both the protein and the solvent and is thelocalized diffusion mode. This slow relaxation enters the HFBStime window only in the case of the hydrated protein but can beseen by DS in both the hydrated and the glycerol solvatedsystems. This slow relaxation is a strongly protein-solventcoupled relaxation, and thus its temperature dependence iscontrolled by the solvent viscosity (which is the lowest for water).The coupling mechanism is in agreement with a picture ofsolvent molecules interacting well with the protein surface.
Analysis of the DS data shows a smooth temperaturedependence of the characteristic relaxation time and no sign ofa dynamical transition4,25 (see also Fig. S2 in the ESI†). Thisresult emphasizes that the dynamic transition observed as asharp increase in MSD is caused by the slow process enteringthe resolution window of the HFBS spectrometer. This point canbe well illustrated using the neutron scattering susceptibilityplot in a broad frequency range (Fig. 4). The spectra of WL at T�295 K show a clear peak at n � 6 GHz that indicates the slowprocess.4,25 Spectra of other samples show much weaker peaksin the same frequency range. Similarity of HFBS spectrabetween DL and LT conrms that the relaxation of LT can beascribed to methyl dynamics which dominate the spectra of drylysozyme.21–24 However, the spectra of LG at T ¼ 320 K show atail of the slower processes that enters the resolution windowonly at low frequencies. Using this spectrum, we estimated thecharacteristic frequency of the peak to be n � 0.06 GHz (fordetails see the ESI†). This estimate agrees well with the dielec-tric spectroscopy results (Fig. S2 in ESI†) and suggests that theslow processes in proteins follow well the dynamics of solventglycerol. In fact the LG relaxation time is around �2 orders ofmagnitude slower than that in water (0.06 GH at 320 K vs. 6 GHzat 295 K) and the viscosity of bulk glycerol is around 300 timeshigher than bulk water at room temperature. The LT spectra atlower frequencies are dominated by the methyl dynamics(Fig. 4). This is consistent with the absence of the dynamictransition in MSD of LT up to this temperature.
Fig. 4 Combined HFBS and DCS c0 0(n) spectra of DL, WL, LG and LT at ambienttemperature. The solid line shows a fit of the peak in WL by a Cole–Cole functionwith the stretching parameter a � 0.2 (for details see the ESI†).
This journal is ª The Royal Society of Chemistry 2013
Conclusions
In understanding the efficacy of bioprotectants, we show thatfast conformational uctuations of the protein might be the keyprecursors to the larger structural and global protein motions,and that in choosing the best storage formulation with glassformers, one needs to consider the interplay between fragilityand Tg. If the viscosity of the solvent becomes lower, the slowrelaxation process is already active and becomes the dominantfactor for long-term stability. As a result, at room temperature orabove, trehalose helps to suppress slower modes and improvesprotein stability. It is the high Tg of trehalose that makes itbetter at high T. The data suggest that trehalose molecules formhydrogen bonds with the protein surface, replacing the watermolecules, and form a “glassy crust” suppressing proteindynamics at high T. This is in accordance with the existinghypothesis of glass stabilization and gives some insight into theeffectiveness of trehalose as the ‘magic’ sugar for preservation,in line with some known organisms being able to survive in atrehalose environment in the absence of water at high T.33
However, at low temperatures, the data point to glycerol asthe better cryogenic solvent. It packs efficiently and forms astrong glass integrated with protein residues, thus suppressingfast motions in the glassy state. In contrast, trehalose is a fragileglass, packs less efficiently and is not able to interact tightlywith the protein surface, only forming larger cages and allowingfor slightly enhancedmobility. Our neutron scattering results inlow temperature regions contradict the previous idea thattrehalose is the most efficient biopreserving agent due to itshigh Tg. There are liquids (e.g. glycerol) that might be moreefficient at low temperatures, although they have lower Tg.
Notes and references
1 J. H. Crowe, L. M. Crowe and D. Chapman, Science, 1984, 223,701.
2 K. B. Storey and J. M. Storey, Sci. Am., 1990, 263, 92.3 W. Doster, S. Cusack and W. Petry, Nature, 1989, 337,754.
4 S. Khodadadi, S. Pawlus, J. H. Roh, V. Garcıa Sakai,E. Mamontov and A. P. Sokolov, J. Chem. Phys., 2008, 128,195106.
5 S. Khodadadi, J. H. Roh, A. Kisliuk, E. Mamontov, M. Tyagi,S. A. Woodson, R. M. Briber and A. P. Sokolov, Biophys. J.,2010, 98, 1321.
6 S.-H. Chen, L. Liu, P. Baglioni, A. Faraone and E. Mamontov,Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 9012.
7 L. Cordone, M. Ferrand, E. Vitrano and G. Zaccai, Biophys. J.,1999, 76, 1043.
8 J. H. Roh, M. Tyagi, R. M. Briber, S. A. Woodson andA. P. Sokolov, J. Am. Chem. Soc., 2011, 133, 16406.
9 A. Paciaroni, S. Cinelli and G. Onori, Biophys. J., 2002, 83,1157.
10 T. E. Dirama, J. E. Curtis, G. A. Carri and A. P. Sokolov,J. Chem. Phys., 2006, 124, 034901.
11 T. E. Dirama, G. A. Carri and A. P. Sokolov, J. Chem. Phys.,2005, 122, 244910.
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12 G. Caliskan, D. Mechtani, J. H. Roh, A. Kisliuk, A. P. Sokolov,S. Azzam, M. T. Cicerone, S. Lin-Gibson and I. Peral, J. Chem.Phys., 2004, 121, 1978.
13 A. L. Tournier, J. Xu and J. C. Smith, Biophys. J., 2003, 85,1871.
14 A. de Gusseme, L. Carpentier, J. F. Willart and M. Descamps,J. Phys. Chem. B, 2003, 107, 10879.
15 N. K. Jain and I. Roy, Protein Sci., 2009, 18, 24.16 M. T. Cicerone, A. Tellington, L. Trost and A. P. Sokolov,
BioProcess Int., 2003, 1, 36.17 C. A. Angell, Science, 1995, 267, 1924.18 M. T. Cicerone and C. L. Soles, Biophys. J., 2004, 86,
3836.19 G. Madhavi Sastry and N. Agmon, Biochemistry, 1997, 36,
7097.20 S. Khodadadi, J. E. Curtis and A. P. Sokolov, J. Phys. Chem. B,
2011, 115, 6222.21 J. H. Roh, J. E. Curtis, S. Azzam, V. K. Novikov, I. Peral,
Z. Chowdhuri, R. B. Gregory and A. P. Sokolov, Biophys. J.,2006, 91, 2573.
22 J. E. Curtis, T. E. Dirama, G. A. Carri and D. J. Tobias, J. Phys.Chem. B, 2006, 110, 22953.
5340 | Soft Matter, 2013, 9, 5336–5340
23 J.H.Roh,V.N.Novikov,R.B.Gregory, J. E.Curtis, Z.Chowdhuriand A. P. Sokolov, Phys. Rev. Lett., 2005, 95, 038101.
24 W. Doster, Biochim. Biophys. Acta, Proteins Proteomics, 2010,1804, 3.
25 S. Khodadadi, S. Pawlus and A. P. Sokolov, J. Phys. Chem. B,2008, 112, 14273.
26 R. B. Gregory, The Role of Water in Foods, Chapman-Hall,New York, 1998, p. 57.
27 D. Averett, M. T. Cicerone, J. F. Douglas and J. de Pablo, SoMatter, 2012, 8, 4936.
28 A. P. Sokolov, E. Rossler, A. Kisliuk and D. Quitmann, Phys.Rev. Lett., 1993, 71, 2062.
29 A. M. Tsai, D. A.Neumann and L. N. Bell, Biophys. J., 2000, 79,2728.
30 E. Mamontov, H. O’Neill and Q. Zhang, J. Biol. Phys., 2010,36, 291.
31 G. Caliskan, A. Kisliuk, A. M. Tsai, C. L. Soles andA. P. Sokolov, J. Chem. Phys., 2003, 118, 4230.
32 L. Hong, N. Smolin, B. Lindner, A. P. Sokolov and J. C. Smith,Phys. Rev. Lett., 2011, 107, 148102.
33 J. H. Crowe, J. F. Carpenter and L. M. Crowe, Annu. Rev.Physiol., 1993, 60, 73.
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