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Concepts and problems in protein dynamics
Chemical Physics 424 (2013) 2–6
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ChemPhys Perspective
Concepts and problems in protein dynamics Paul W. Fenimore a,⇑, Hans Frauenfelder a, Salvatore Magazù b, Benjamin H. McMahon a, Ferenc Mezei c, Federica Migliardo b, Robert D. Young d, Izabela Stroe e a
Los Alamos National Laboratory, Los Alamos, NM 87545, USA Department of Physics and Earth Sciences, University of Messina, 98166 Messina, Italy c European Spallation Source ESS AB, Lund, Sweden d Arizona State University, Tempe, AZ 85287, USA e Worcester Polytechnic Institute, Worcester, MA 01609, USA b
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Article history: Available online 25 July 2013 Keywords: Protein dynamics Neutron scattering Mössbauer spectroscopy
a b s t r a c t The function of proteins depends crucially on conformational motions. The characteristic times of these motions extend from sub-picosecond to seconds. No single experimental tool can cover the entire time range and provide all necessary parameters for a complete understanding. Moreover, without a solid understanding of the data evaluation it is easy to misinterpret the complex phenomena. Because protein motions are truly complex, the evaluation of the data even from such well-known techniques as neutron scattering (Magazu and Migliardo, 2011 [1]) and the Mössbauer effect (Chen and Yang, 2007 [2]) can lead to erroneous concepts and conclusions. We believe that notions such as the Lamb–Mössbauer relation, the protein dynamic transition, the protein glass transition, and the dynamic crossover are misleading or misapplied. To justify this statement we first briefly describe our view of dynamic proteins and then explain why we believe that these notions should be revised or abandoned. Ó 2013 Elsevier B.V. All rights reserved.
1. Concepts of protein dynamics The universe of proteins is enormous. The number of different proteins exceeds a million by many orders of magnitude [3]. Studying one protein in depth takes at least a year. It is thus impossible to study even a very small fraction of all proteins. Needed are concepts that apply to all biomolecules. We believe that the following concepts are necessary to understand protein dynamics, namely conformational substates, the free-energy landscape, fluctuations, and the control of internal protein motions by external fluctuations. We have explained these concepts in earlier publications in detail and only sketch them here. We then use these well-established concepts to justify our belief that some notions in general use are misleading or wrong. 1.1. Conformational substates Proteins do not exist in a unique conformation as is implied by textbook figures; they can assume an extremely large number of different conformations or conformational substates. This fact was experimentally found by showing that non-exponential rebinding in flash photolysis experiments could only be explained by assuming proteins to be inhomogeneous [4]. Since these early ⇑ Corresponding author. Tel.: +1 505 665 7744. E-mail address: [email protected] (P.W. Fenimore). 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2013.06.023
experiments the concept of substates has been confirmed by additional experiments and by simulations [5]. Most convincingly, thermodynamics requires the existence of substates [6]. In a protein ensemble no two proteins are identical at any given time. Without the existence of the conformational substates, proteins would be like diamonds, beautiful and sparkling, but lifeless. 1.2. The free-energy landscape The structure of a given protein in a particular substate can in principle be described by giving the coordinates of all atoms. The structure can thus be considered a point in the very-high dimensional conformational energy landscape. A map of the free-energy of a protein for all substates gives the free-energy landscape. It is of course impossible to draw this landscape, but two dimensional cross sections through the landscape give at least some idea of the complexity of proteins [7]. 1.3. Fluctuations The existence of substates alone does not make proteins fit for life. In order to perform functions they must move from substate to substate. Protein motions are dominated by three types of fluctuations, a, bh, and vibrations. These fluctuations are known from the physics of glasses and supercooled liquids [8–10]. The a fluctuations largely control protein shape changes [11]. Their rate
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coefficient is inversely proportional to the solvent viscosity; their temperature dependence is usually approximated by the Vogel– Fulcher–Tammann relation. The bh fluctuations are largely due to the hydration shell of the proteins [12]; their temperature dependence follows an Arrhenius-type law. Fig. 1 gives the temperature dependence of the two types of fluctuations in a 1/1 glycerol– water sample. The number of vibrations in a protein is very large and can only in some cases be described individually. To understand the role of the fluctuations in protein dynamics, the temperature dependence of a single rate coefficient alone is not sufficient. The frequency spectra are equally important because they determine the probability distribution of the fluctuations (a density of states). The spectra of the a fluctuations are symmetric and narrower than the asymmetric spectra of the bh fluctuations. Fig. 2 displays the spectrum of the bh fluctuations in myoglobin embedded in a hydrated poly(vinyl alcohol) glass taken at several temperatures. Also shown for a later discussion are three vertical lines characterizing three rate coefficients, namely for the emission of the 57Fe 14.4 keV gamma rays, the energy resolution of the neutron scattering instrument IN13, and the rate coefficient of a typical differential scanning calorimetry instrument. In a typical protein–solvent sample, the a fluctuations originate in the bulk solvent. They are absent or too slow to be important if the protein is embedded in a high-viscosity environment, such as a crystal. The b fluctuations can be associated with small ice crystals in the bulk solvent or be associated with the hydration shell. The bh fluctuations in the hydration shell depend on the degree of hydration and are absent if the protein is dehydrated [13,14]. The fact that a fluctuations are absent in high viscosity solvents and bh fluctuations are absent in dehydrated proteins permits one to study the effect of these fluctuations on protein dynamics and function separately. At present, the observed phenomena can be understood on the basis of the two types of fluctuations, a and bh. Additional fluctuation processes exist [15–17], but their functional roles are not yet clear.
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Fig. 2. The spectra of the bh fluctuations in the hydration shell of myoglobin embedded in solid poly(vinyl alcohol), with hydration h = 0.4 g/g. The vertical lines give the values for the rate coefficients characterizing differential scanning calorimetry, the Mössbauer effect in 57Fe, and for neutron scattering (IN13). Spectra are measured by dielectric relaxation spectroscopy.
After this overview, we look at some common notions that are used to describe protein dynamics and ask if they are correct, or if a reevaluation is needed. 2. Debye–Waller and the Lamb–Mössbauer factors Two relations are frequently used to evaluate the experiments that explore protein structure and dynamics, namely the Debye– Waller factor (DWF) and the Lamb–Mössbauer factor (LMF). Both are usually written as a temperature-dependent fraction
fðTÞ ¼ exp q2 < x2 > :
ð1Þ
1.4. Important protein motions are externally controlled Proteins would be useless in living systems if they could not be influenced by external agents. Experiments indeed show that for instance the viscosity of the surroundings and the degree of hydration influence protein dynamics [14,18–20]. The a fluctuations change the shape of the protein and for instance open and close the entrance and exit to the heme pocket in myoglobin. The bh fluctuations in the hydration shell control some internal channels.
Fig. 1. Temperature dependence of the a and bh fluctuations in a 1/1 glycerol/water sample.
Here f(T) is an elastic fraction, q is a wave vector and a meansquared displacement (msd). In X-ray crystallography f(T) is called the Debye–Waller factor. It gives the reduction in coherent scattering intensity because the scattering centers are not in unique positions, but are distributed in volumes characterized by the msd [21]. The msd can be decomposed into different components, usually vibrational and conformational [22–24]. Eq. (1) is only valid for harmonic and isotropic motions. This limitation is significant when conformational motions contribute to the variation in atomic positions between unit cells [25]. Nevertheless, the Debye–Waller factor is routinely used in the studies of biomolecular structure and provides useful information. In incoherent neutron scattering and in Mössbauer experiments Eq. (1) is called the Lamb–Mössbauer relation. Its application to proteins leads to problems. The scattered radiation appears to show a sharp line and a broad band. The sharp line is taken to describe the elastic scattering, the broad band the quasi-elastic scattering. The two components are then separated as cleanly as possible and treated individually. The fraction f(T) of the sharp line is assumed to be given by Eq. (1). The fraction 1 f(T) is denoted as quasi-elastic scattering and is usually assumed to describe diffusional motion of the scatterer. Theseparation into two different components is, in our opinion, wrong [26]. The Doppler broadening is caused by external fluctuations that produce sharp sidebands as will be discussed in more detail below. The entire spectrum consists of an extremely large number of sharp lines. In an earlier paper [27] we showed that the entire Mössbauer spectrum is caused by the bh fluctuations. The separation into a sharp elastic line and a broad quasi-elastic band is wrong. The msd in the
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Lamb–Mössbauer relation should be discarded and data should be described by the measured fraction f(T) and not by the msd [14].
3. The putative protein dynamical transition Above about 200 K hydrated proteins show a dramatic increase of their msd. The effect was first seen in Mössbauer experiments [28–31]. Later it was also observed in neutron scattering and baptized the ‘‘protein dynamical transition’’ or PDT [32]. The PDT has been seen in many proteins [33] and has been declared a generic property of proteins [34]. The PDT has led to many discussions and to many experiments designed to elucidate its nature. The proposed explanations fall into two general categories: I. When the relaxation time of the system intersects the instrumental response time (or the corresponding energy resolution), motions become visible to the instrument, giving a larger measured msd [14,35– 37]. II. In the conventional model, the PDT is explained by a shift in thermodynamic occupation or strongly-temperature dependent change in the intrinsic dynamics, exemplified in the simplest case by jumps between two unequal wells separated by a distance d and free energy DG = DH TDS. The change in these dynamics with temperature is typically blamed on a glass-like transition in the hydration shell [29–31,33,38]. A successful model must explain all experimentally observed features and must have predictive power. The essential features to be explained are the absence of the PDT in dehydrated systems, the temperature dependence of the msd, and the shapes of the broad lines seen in the Mössbauer effect and the incoherent neutron scattering. The analysis of neutron scattering data with a detailed accounting for instrumental energy resolution is consistent with Model I, and requires no elements of Model II [35,36,39]. As illuminating as this analysis is, it does not predict either the temperature-dependent msd, nor the lineshapes. Analysis postulating Model II fails because there is no unique evidence for a two-well landscape in proteins and because the parameters describing the wells are not predicted, but must be obtained at each temperature by data fitting. The lineshapes are not predicted because there is typically no frequency-resolved data available as input. The ‘‘unified model’’ originated by realizing that the PDT can be caused by fluctuations in the hydration shell and the bulk solvent [14,37]. Measurements of dielectric response provided an independent and frequency-resolved window into bulk and hydration water dynamics. The distributions measured by dielectric response spectroscopy allowed the unified model to predict the onset and shape of f(T) without free parameters using the dielectric relaxation spectroscopy measured shape of the bh fluctuations [14]. This fact can be understood by looking at Fig. 2. The shape of the bh spectrum is essentially temperature independent. At temperatures well below 200 K all rate coefficients that form the spectrum are slower than the rate coefficient kMo characteristic of the resolution of the Mössbauer effect. As the temperature is increased the spectrum shifts to higher frequencies. At about 200 K, its high-frequency wing reaches kMo. The conformational motions produced by the bh fluctuations shift some of the components of the sharp Mössbauer line to higher and some to lower energies; thus f(T) decreases and the msd increases. The quantitative analysis yields the temperature dependence of the msd for both Mössbauer and neutron without adjustable parameters [14,40]. This model proves that the PDT is no mystery; it is quantitatively explained as being caused by the dielectric fluctuations in the hydration shell. The model also explains the full Mössbauer spectrum by using the theory of Singwi and Sjölander [41] and one dimensionless fitting parameter [27]. The unified model is strengthened by ancient data. In 1960, Ruby and Bolef acoustically modulated a 57Fe absorber with an ultrasound of frequency t = 2 107 s1 [42]. The ensuing
spectra showed a decreased central line and sidebands separated by energies ht. Thus the PDT is caused by shifting sharp Mössbauer lines into the sideband. As an aside we mention an often cited paper by Lichtenegger et al. [43] which shows the temperature dependence of the msd of 57Fe embedded in two solvents that differ in viscosity by more than a factor 107. The onset of the PDT in the two samples is shifted by only 20 K. The authors explain the result by postulating that the viscosity at the protein surface is much smaller than in the bulk, a feature that is difficult to accept. The bh fluctuations explain the result convincingly. The two solvents have similar hydrations and thus presumably similar fluctuations. The hydration is somewhat smaller for the sample with higher viscosity, which can account for the shift of the kink. Model independent neutron scattering spectroscopic surveys showed that the increase of the fluctuations with temperature (as predicted by the unified model) within a given rate window is indeed the origin of the increased msd values obtained by model fitting [44]. 4. The protein glass transition The distinction between Models I and II is not absolute. One can imagine theories that involve both rate processes moving through an instrumental observation window, and some change in thermodynamic weights. Differential scanning calorimetry (DSC) shows an exceptionally broad relaxation process in some hydrated proteins [33,45–47]. The nature of this process, sometimes called the ‘‘Protein Glass Transition’’ (PGT), remained a puzzle for many years. The PGT phenomenology measured using DSC is also explained by the unified model [14] as a result of solvent a and hydration bh fluctuations [48]. The effect is illustrated with Fig. 2. The instrumental rate coefficient kDSC for DSC is about 10 s1. At 150 K, all kb are slower than kDSC and no anomaly in differential temperature is measured. At 200 K some kb are faster than kDSC, leading to the observed increase in the heat capacity and the phenomenology of the PGT. At 295 K, all fluctuations are much faster than kDSC and the PGT is completed. Both, a and b fluctuations, can cause the ‘‘protein glass transition’’. Thus the ‘‘Protein Dynamic Transition’’ and the ‘‘Protein Glass Transition’’ have the same origin, namely external fluctuations. The difference between the two is due to different instrumental rate coefficients that determine the outcome of the measurement. 5. Does the dynamical crossover exist? Some dielectric relaxation spectra of proteins have been interpreted as showing that low-temperature b fluctuations change into a fluctuations at about 200 K [49–52]. This phenomenon is called ‘‘dynamical crossover’’. The effect, if correct, is surprising because a and bh have different origins and different roles in protein dynamics. The a fluctuations are caused by the bulk solvent, are inversely proportional to the solvent viscosity, and control the shape of proteins [11]. The bh fluctuations originate partially or fully from the hydration shell. They are essentially independent of viscosity, depend on hydration, and control some internal fluctuations. These features make the existence of a ‘‘dynamical crossover’’ difficult to accept. Indeed, the crossing of a and bh fluctuations has been observed, for instance in [53] and also in our experiments, shown in Fig. 1. We believe that the apparent crossover is caused by the data evaluation. In the case of two broadly distributed rate processes crossing on an Arrhenius plot (i.e. the frequency-resolved, temperature-dependent dielectric loss spectrum of a and bh), it can be difficult to separate the two processes at a single temperature by assigning a fraction of the observed distribution to the two processes. Only by including a model of the temperature
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dependence of a and bh can a stable decomposition be obtained at their crossing, near 215 K [14]. Certainty that the processes cross comes only from the hydration and temperature dependence of bh, and the consistency of a’s temperature dependence between samples with and without protein (i.e. with and without bh). Unfortunately, many other papers do not provide enough information about their data evaluation procedure to support or reject this explanation of the dynamical crossover for their data. 6. Conclusions Neutron scattering and the Mössbauer effect yield significant data for protein dynamics, but a number of conceptual problems exist. Arguments, sometimes heated, have persisted for a long time. We hope that the present paper leads to reasoned discussion of the disputed questions so that the results from these techniques can be used with confidence. Acknowledgments We thank Joel Berendzen, Jason Lashley, and Dean Taylor for intense discussions that have been important for understanding the problems and difficulties in protein dynamics. References [1] S. Magazu, F. Migliardo, Dynamics of Biological Macromolecules by Neutron Scattering. Benthamscience eBook, 2011. [2] Y.-L. Chen, D.-P. Yang, Mössbauer Effect in Lattice Dynamics, Wiley-VCH, Weinheim, 2007. [3] R.M. May, PLoS Biol. 9 (2011) 8. [4] R.H. Austin, K.W. Beeson, L. Eisenstein, H. Frauenfelder, I.C. Gunsalus, Biochemistry 14 (1975) 5355. [5] H. Frauenfelder, F. Parak, R.D. Young, Annu. Rev. Biophys. Biophys. Chem. 17 (1988) 451. [6] A. Cooper, Prog. Biophys. Mol. Biol. 44 (1984) 181. [7] H. Frauenfelder, S.G. Sligar, P.G. Wolynes, Science 254 (1991) 1598. [8] F. Kremer, A. Schönhals, Broadband Dielectric Spectroscopy, Springer, 2003. [9] P.G. Wolynes, V. Lubchenko, Structural Glasses and Supercooled Liquids, Wiley, 2012. [10] K.L. Ngai, Relaxation and Diffusion in Complex Systems, Springer, 2011. [11] V. Lubchenko, P.G. Wolynes, H. Frauenfelder, J. Phys. Chem. B 109 (2005) 7488. [12] D.N. LeBard, D.V. Matyushov, J. Phys. Chem. B 114 (2010) 9246. [13] L. Zhang, L. Wang, Y.-T. Kao, W. Qiu, Y. Yang, O. Okobiah, D. Zhong, PNAS 104 (2007) 18461. [14] H. Frauenfelder, G. Chen, J. Berendzen, P.W. Fenimore, H. Jansson, B.H. McMahon, I.R. Stroe, J. Swenson, R.D. Young, Proc. Natl. Acad. Sci. 106 (2009) 5129. [15] J.G.P. Singh, F. Parak, S. Hunklinger, K. Dransfeld, Phys. Rev. Lett. 47 (1981) 685. [16] S.A. Lusceac, M.R. Vogel, C.R. Herbers, Biochim. Biophys. Acta 1804 (2010) 41. [17] L. Hong, N. Smolin, B. Lindner, A.P. Sokolov, J.C. Smith, Phys. Rev. Lett. 107 (2011) 148102. [18] D. Beece, L. Eisenstein, H. Frauenfelder, D. Good, M.C. Marden, L. Reinisch, A.H. Reynolds, L.B. Sorensen, K.T. Yue, Biochemisty 19 (1980) 5147. [19] T. Kleinert, W. Doster, H. Leyser, W. Petry, V. Schwarz, M. Settles, Biochemistry 37 (1998) 717. [20] H. Frauenfelder, P.W. Fenimore, B.H. McMahon, Biophys. Chem. 98 (2002) 35. [21] B.T.M. Willis, A.W. Pryor, Thermal Vibrations in Crystallography, Cambridge University Press, 1975. [22] H. Frauenfelder, G.A. Petsko, D. Tsernoglou, Nature 280 (1979) 558. [23] G.A. Petsko, D. Ringe, Ann. Rev. Biophys. Bioeng. 13 (1984) 331. [24] L. Maragliano, G. Cottone, L. Cordone, G. Ciccotti, Biophys. J. 86 (2004) 2765. [25] A.E. Garcia, J.A. Krumhansl, H. Frauenfelder, Proteins 29 (1997) 153. [26] H. Frauenfelder, R.D. Young, P.W. Fenimore, J. Phys. Chem. B, submitted for publication. [27] R.D. Young, H. Frauenfelder, P.W. Fenimore, Phys. Rev. Lett. 107 (2011) 158102. [28] F. Parak, H. Formanek, Acta Cryst. A 27 (1971) 573. [29] H. Keller, P.G. Debrunner, Phys. Rev. Lett. 45 (1980) 68. [30] S.G. Cohen, E.R. Bauminger, I. Nowik, S. Ofer, J. Yariv, Phys. Rev. Lett. 46 (1981) 1244. [31] F. Parak, E.N. Frolov, R.L. Mössbauer, V.I. Goldanskii, J. Mol. Biol. 145 (1981) 825. [32] W. Doster, S. Cusack, W. Petry, Nature 337 (1989) 754. [33] W. Doster, Biochim. Biophys. Acta 1804 (2010) 3. [34] W. Doster, S. Busch, A.M. Gaspar, M.-S. Appavou, J. Wuttke, H. Scheer, Phys. Rev. Lett. 104 (2010) 098101. [35] S. Magazu, F. Migliardo, A. Benedetto, J. Phys. Chem. B 115 (2011) 7736.
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Dr. Paul Fenimore received a B.S. from the Physics Department at Rensselaer Polytechnic Institute, and a Ph.D. from the Physics Department at the University of Illinois, Urbana-Champaign, He came to Los Alamos National Laboratory as a post-doc in 2001, and since 2004 he has been a staff scientist in the Theoretical Biology and Biophysics Group at Los Alamos National Laboratory. His published research includes papers on protein dynamics, function, and their coupling to solvent and hydration, mathematical epidemiology, and vaccine design. He is a recipient of two Los Alamos Small Team Distinguished Performance Awards.
Hans Frauenfelder received his Ph. D. in physics in 1950 from the Swiss Federal Institute of Technology. In 1952 he moved to the Physics Department of the University of Illinois. From 1992 to 2012 he was in the theory division of the Los Alamos National Laboratory. His research ranged from perturbed angular correlation to the Mössbauer effect and parity violation in nuclear physics. His current interest is protein dynamics.
Salvatore Magazù is Full Professor in Experimental Physics and Head of the research group in Structure of Matter and Biophysics at the Physics and Earth Sciences Department of Messina University, Italy. He is president of the Le Studium International Consortium COSMO (France) and of the Interuniversitary Consortium for Applied Physical Sciences CISFA (Italy); he has been associated researcher of Le Studium, Institute for Advanced Studies, at the laboratory Conditions Extrêmes et Matériaux : Haute Température et Irradiation CEMHTI – CNRS in Orléans; chairman and member of scientific committees at the European Synchrotron Radiation Facility and at the Institut Laue-Langevin in Grenoble. His research topics are manifold and, from some points of view, different, even if it emerges a common thread: the integrated employment of spectroscopic techniques, such as neutron scattering, synchrotron radiation spectroscopy and light scattering, addressed to characterize the structural and dynamical properties of systems of biophysical interest. He is author of more than 300 articles in international journals and more than 200 communications in conferences. He has received several international and national awards, such as the 2000 Scientia Europaea Prize by the French Academy of Sciences and Aventis.
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P.W. Fenimore et al. / Chemical Physics 424 (2013) 2–6 Ben McMahon is Deputy Group Leader in the Theoretical Biology and Biophysics Group at Los Alamos National Laboratory. His research interests range from the physical chemistry of protein dynamics and function to evolutionary analysis to systems-level modeling, such as epidemiology or disease progression.
Ferenc Mezei graduated with a Physics Diploma from Eötvös L. University, Budapest in 1965, and a Ph.D. from the Hungarian Academy of Sciences, Budapest in 1976. He has worked at Eötvös L. University, the Central Research Institute for Physics, Budapest, the InstitutLaue-Langevin, was professor at the Technical University, Berlin from 1984-2007, Director of the Berlin Neutron Scattering Center at the Hahn-Meitner-Institut, Berlin from 1992-1997 and 2001-2007, and at Los Alamos National Laboratory from 1997-2010, first as the John Wheatley Scholar and then as a Technical Staff Member from 2000-2010. He is an Ordinary Member of the Hungarian Academy of Sciences, a member of Academia Europaea, and former Chairman of the Action Committee on Publications of the European Physical Society 1989 – 1994. He has been awarded the Janossy Prize (1979), the Hewlett-Packard Europhysics Prize (1968), the Leo Szilard Medal (1996), the Middle Cross of the Republic of Hungary (1997), the Inaugural Walter Haelg Prize, and is a Fellow of the American Physical Society. He invented and developed neutron spin-echo and neutron supermirrors, provided experimental proof of the Landau-Khalatnikov relation for rotons in He, discovered the break-down of dynamic scaling in ferromagnets and dynamic scaling near glass transitions, and invented long pulse neutron sources, ballistic neutron guides, and multiplexing chopper system for pulsed spallation sources. He is currently the Head of the Target Division of the European Spallation Source and an adjunct professor at the University of California San Diego,.
Federica Migliardo is a biophysicist at the Department of Physics and Earth Sciences of the University of Messina, Italy. She has been ATER (Attaché Temporaire d’Enseignement et de Recherche) and UNESCO-L’Oréal International Fellow at the Laboratoire de Dynamique et Structure des Matériaux Moléculaires of the University of Lille 1 and European Molecular Biology Organisation (EMBO) International Fellow at the Institut de Biochimie et Biophysique Moléculaire et Cellulaire of the University of Paris-Sud XI, France. Her research activity is focused on the study of the molecular mechanisms responsible for biological processes, such as bioprotection, denaturation and stabilisation of biomolecules and, more recently, for infective (tuberculosis and schistosomiasis) and neurodegenerative (Parkinson) diseases. Her
methodological approach is based on the synergistic application of complementary techniques allowing to access to different space and time domains, in particular neutron and light scattering. For her research achievements, testified by more than 150 articles, she received several awards, such as, more recently, the International BioVision Award for Life Sciences 2011, the UNESCO-L’Oréal For Women in Science International Fellowship 2008 and the European EUWIIN Special Recognition Award 2007.
Robert D. Young is a physicist. He is currently adjunct faculty at Arizona State University. Prior to that, he was a professor and administrator at Illinois State University. He was also adjunct research professor at the University of Illinois at Champaign-Urbana. Early on, he specialized in elementary particle theory. In mid-career, biological physics became his consuming interest. He works on building dynamic models of proteins with environment included, based on fluctuations in their complex energy landscape. He currently resides in Flagstaff, Arizona, and enjoys being outside in the Grand Canyon of the Colorado River.
Izabela R. Stroe received her B.Sc. from the University of Bucharest in 1994 and her Ph.D. in Physics from Clark University in 2005. She joined the Los Alamos National Laboratory as a Seaborg Postdoctoral Fellow under the guidance of Dr. Albert Migliori working on the properties of actinides, correlated electrons systems and biological systems. In 2008 she joined the Physics Department at Worcester Polytechnic Institute as assistant professor tenure track. Her research aims at understanding the protein-solvent coupling using complementary experimental tools. She is a recipient of the Kalenian Award for innovation and entrepreneurship for her work on early detection of amyloidogenic precursors.
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ChemPhys Perspective
Concepts and problems in protein dynamics Paul W. Fenimore a,⇑, Hans Frauenfelder a, Salvatore Magazù b, Benjamin H. McMahon a, Ferenc Mezei c, Federica Migliardo b, Robert D. Young d, Izabela Stroe e a
Los Alamos National Laboratory, Los Alamos, NM 87545, USA Department of Physics and Earth Sciences, University of Messina, 98166 Messina, Italy c European Spallation Source ESS AB, Lund, Sweden d Arizona State University, Tempe, AZ 85287, USA e Worcester Polytechnic Institute, Worcester, MA 01609, USA b
a r t i c l e
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Article history: Available online 25 July 2013 Keywords: Protein dynamics Neutron scattering Mössbauer spectroscopy
a b s t r a c t The function of proteins depends crucially on conformational motions. The characteristic times of these motions extend from sub-picosecond to seconds. No single experimental tool can cover the entire time range and provide all necessary parameters for a complete understanding. Moreover, without a solid understanding of the data evaluation it is easy to misinterpret the complex phenomena. Because protein motions are truly complex, the evaluation of the data even from such well-known techniques as neutron scattering (Magazu and Migliardo, 2011 [1]) and the Mössbauer effect (Chen and Yang, 2007 [2]) can lead to erroneous concepts and conclusions. We believe that notions such as the Lamb–Mössbauer relation, the protein dynamic transition, the protein glass transition, and the dynamic crossover are misleading or misapplied. To justify this statement we first briefly describe our view of dynamic proteins and then explain why we believe that these notions should be revised or abandoned. Ó 2013 Elsevier B.V. All rights reserved.
1. Concepts of protein dynamics The universe of proteins is enormous. The number of different proteins exceeds a million by many orders of magnitude [3]. Studying one protein in depth takes at least a year. It is thus impossible to study even a very small fraction of all proteins. Needed are concepts that apply to all biomolecules. We believe that the following concepts are necessary to understand protein dynamics, namely conformational substates, the free-energy landscape, fluctuations, and the control of internal protein motions by external fluctuations. We have explained these concepts in earlier publications in detail and only sketch them here. We then use these well-established concepts to justify our belief that some notions in general use are misleading or wrong. 1.1. Conformational substates Proteins do not exist in a unique conformation as is implied by textbook figures; they can assume an extremely large number of different conformations or conformational substates. This fact was experimentally found by showing that non-exponential rebinding in flash photolysis experiments could only be explained by assuming proteins to be inhomogeneous [4]. Since these early ⇑ Corresponding author. Tel.: +1 505 665 7744. E-mail address: [email protected] (P.W. Fenimore). 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2013.06.023
experiments the concept of substates has been confirmed by additional experiments and by simulations [5]. Most convincingly, thermodynamics requires the existence of substates [6]. In a protein ensemble no two proteins are identical at any given time. Without the existence of the conformational substates, proteins would be like diamonds, beautiful and sparkling, but lifeless. 1.2. The free-energy landscape The structure of a given protein in a particular substate can in principle be described by giving the coordinates of all atoms. The structure can thus be considered a point in the very-high dimensional conformational energy landscape. A map of the free-energy of a protein for all substates gives the free-energy landscape. It is of course impossible to draw this landscape, but two dimensional cross sections through the landscape give at least some idea of the complexity of proteins [7]. 1.3. Fluctuations The existence of substates alone does not make proteins fit for life. In order to perform functions they must move from substate to substate. Protein motions are dominated by three types of fluctuations, a, bh, and vibrations. These fluctuations are known from the physics of glasses and supercooled liquids [8–10]. The a fluctuations largely control protein shape changes [11]. Their rate
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coefficient is inversely proportional to the solvent viscosity; their temperature dependence is usually approximated by the Vogel– Fulcher–Tammann relation. The bh fluctuations are largely due to the hydration shell of the proteins [12]; their temperature dependence follows an Arrhenius-type law. Fig. 1 gives the temperature dependence of the two types of fluctuations in a 1/1 glycerol– water sample. The number of vibrations in a protein is very large and can only in some cases be described individually. To understand the role of the fluctuations in protein dynamics, the temperature dependence of a single rate coefficient alone is not sufficient. The frequency spectra are equally important because they determine the probability distribution of the fluctuations (a density of states). The spectra of the a fluctuations are symmetric and narrower than the asymmetric spectra of the bh fluctuations. Fig. 2 displays the spectrum of the bh fluctuations in myoglobin embedded in a hydrated poly(vinyl alcohol) glass taken at several temperatures. Also shown for a later discussion are three vertical lines characterizing three rate coefficients, namely for the emission of the 57Fe 14.4 keV gamma rays, the energy resolution of the neutron scattering instrument IN13, and the rate coefficient of a typical differential scanning calorimetry instrument. In a typical protein–solvent sample, the a fluctuations originate in the bulk solvent. They are absent or too slow to be important if the protein is embedded in a high-viscosity environment, such as a crystal. The b fluctuations can be associated with small ice crystals in the bulk solvent or be associated with the hydration shell. The bh fluctuations in the hydration shell depend on the degree of hydration and are absent if the protein is dehydrated [13,14]. The fact that a fluctuations are absent in high viscosity solvents and bh fluctuations are absent in dehydrated proteins permits one to study the effect of these fluctuations on protein dynamics and function separately. At present, the observed phenomena can be understood on the basis of the two types of fluctuations, a and bh. Additional fluctuation processes exist [15–17], but their functional roles are not yet clear.
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Fig. 2. The spectra of the bh fluctuations in the hydration shell of myoglobin embedded in solid poly(vinyl alcohol), with hydration h = 0.4 g/g. The vertical lines give the values for the rate coefficients characterizing differential scanning calorimetry, the Mössbauer effect in 57Fe, and for neutron scattering (IN13). Spectra are measured by dielectric relaxation spectroscopy.
After this overview, we look at some common notions that are used to describe protein dynamics and ask if they are correct, or if a reevaluation is needed. 2. Debye–Waller and the Lamb–Mössbauer factors Two relations are frequently used to evaluate the experiments that explore protein structure and dynamics, namely the Debye– Waller factor (DWF) and the Lamb–Mössbauer factor (LMF). Both are usually written as a temperature-dependent fraction
fðTÞ ¼ exp q2 < x2 > :
ð1Þ
1.4. Important protein motions are externally controlled Proteins would be useless in living systems if they could not be influenced by external agents. Experiments indeed show that for instance the viscosity of the surroundings and the degree of hydration influence protein dynamics [14,18–20]. The a fluctuations change the shape of the protein and for instance open and close the entrance and exit to the heme pocket in myoglobin. The bh fluctuations in the hydration shell control some internal channels.
Fig. 1. Temperature dependence of the a and bh fluctuations in a 1/1 glycerol/water sample.
Here f(T) is an elastic fraction, q is a wave vector and
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Lamb–Mössbauer relation should be discarded and data should be described by the measured fraction f(T) and not by the msd [14].
3. The putative protein dynamical transition Above about 200 K hydrated proteins show a dramatic increase of their msd. The effect was first seen in Mössbauer experiments [28–31]. Later it was also observed in neutron scattering and baptized the ‘‘protein dynamical transition’’ or PDT [32]. The PDT has been seen in many proteins [33] and has been declared a generic property of proteins [34]. The PDT has led to many discussions and to many experiments designed to elucidate its nature. The proposed explanations fall into two general categories: I. When the relaxation time of the system intersects the instrumental response time (or the corresponding energy resolution), motions become visible to the instrument, giving a larger measured msd [14,35– 37]. II. In the conventional model, the PDT is explained by a shift in thermodynamic occupation or strongly-temperature dependent change in the intrinsic dynamics, exemplified in the simplest case by jumps between two unequal wells separated by a distance d and free energy DG = DH TDS. The change in these dynamics with temperature is typically blamed on a glass-like transition in the hydration shell [29–31,33,38]. A successful model must explain all experimentally observed features and must have predictive power. The essential features to be explained are the absence of the PDT in dehydrated systems, the temperature dependence of the msd, and the shapes of the broad lines seen in the Mössbauer effect and the incoherent neutron scattering. The analysis of neutron scattering data with a detailed accounting for instrumental energy resolution is consistent with Model I, and requires no elements of Model II [35,36,39]. As illuminating as this analysis is, it does not predict either the temperature-dependent msd, nor the lineshapes. Analysis postulating Model II fails because there is no unique evidence for a two-well landscape in proteins and because the parameters describing the wells are not predicted, but must be obtained at each temperature by data fitting. The lineshapes are not predicted because there is typically no frequency-resolved data available as input. The ‘‘unified model’’ originated by realizing that the PDT can be caused by fluctuations in the hydration shell and the bulk solvent [14,37]. Measurements of dielectric response provided an independent and frequency-resolved window into bulk and hydration water dynamics. The distributions measured by dielectric response spectroscopy allowed the unified model to predict the onset and shape of f(T) without free parameters using the dielectric relaxation spectroscopy measured shape of the bh fluctuations [14]. This fact can be understood by looking at Fig. 2. The shape of the bh spectrum is essentially temperature independent. At temperatures well below 200 K all rate coefficients that form the spectrum are slower than the rate coefficient kMo characteristic of the resolution of the Mössbauer effect. As the temperature is increased the spectrum shifts to higher frequencies. At about 200 K, its high-frequency wing reaches kMo. The conformational motions produced by the bh fluctuations shift some of the components of the sharp Mössbauer line to higher and some to lower energies; thus f(T) decreases and the msd increases. The quantitative analysis yields the temperature dependence of the msd for both Mössbauer and neutron without adjustable parameters [14,40]. This model proves that the PDT is no mystery; it is quantitatively explained as being caused by the dielectric fluctuations in the hydration shell. The model also explains the full Mössbauer spectrum by using the theory of Singwi and Sjölander [41] and one dimensionless fitting parameter [27]. The unified model is strengthened by ancient data. In 1960, Ruby and Bolef acoustically modulated a 57Fe absorber with an ultrasound of frequency t = 2 107 s1 [42]. The ensuing
spectra showed a decreased central line and sidebands separated by energies ht. Thus the PDT is caused by shifting sharp Mössbauer lines into the sideband. As an aside we mention an often cited paper by Lichtenegger et al. [43] which shows the temperature dependence of the msd of 57Fe embedded in two solvents that differ in viscosity by more than a factor 107. The onset of the PDT in the two samples is shifted by only 20 K. The authors explain the result by postulating that the viscosity at the protein surface is much smaller than in the bulk, a feature that is difficult to accept. The bh fluctuations explain the result convincingly. The two solvents have similar hydrations and thus presumably similar fluctuations. The hydration is somewhat smaller for the sample with higher viscosity, which can account for the shift of the kink. Model independent neutron scattering spectroscopic surveys showed that the increase of the fluctuations with temperature (as predicted by the unified model) within a given rate window is indeed the origin of the increased msd values obtained by model fitting [44]. 4. The protein glass transition The distinction between Models I and II is not absolute. One can imagine theories that involve both rate processes moving through an instrumental observation window, and some change in thermodynamic weights. Differential scanning calorimetry (DSC) shows an exceptionally broad relaxation process in some hydrated proteins [33,45–47]. The nature of this process, sometimes called the ‘‘Protein Glass Transition’’ (PGT), remained a puzzle for many years. The PGT phenomenology measured using DSC is also explained by the unified model [14] as a result of solvent a and hydration bh fluctuations [48]. The effect is illustrated with Fig. 2. The instrumental rate coefficient kDSC for DSC is about 10 s1. At 150 K, all kb are slower than kDSC and no anomaly in differential temperature is measured. At 200 K some kb are faster than kDSC, leading to the observed increase in the heat capacity and the phenomenology of the PGT. At 295 K, all fluctuations are much faster than kDSC and the PGT is completed. Both, a and b fluctuations, can cause the ‘‘protein glass transition’’. Thus the ‘‘Protein Dynamic Transition’’ and the ‘‘Protein Glass Transition’’ have the same origin, namely external fluctuations. The difference between the two is due to different instrumental rate coefficients that determine the outcome of the measurement. 5. Does the dynamical crossover exist? Some dielectric relaxation spectra of proteins have been interpreted as showing that low-temperature b fluctuations change into a fluctuations at about 200 K [49–52]. This phenomenon is called ‘‘dynamical crossover’’. The effect, if correct, is surprising because a and bh have different origins and different roles in protein dynamics. The a fluctuations are caused by the bulk solvent, are inversely proportional to the solvent viscosity, and control the shape of proteins [11]. The bh fluctuations originate partially or fully from the hydration shell. They are essentially independent of viscosity, depend on hydration, and control some internal fluctuations. These features make the existence of a ‘‘dynamical crossover’’ difficult to accept. Indeed, the crossing of a and bh fluctuations has been observed, for instance in [53] and also in our experiments, shown in Fig. 1. We believe that the apparent crossover is caused by the data evaluation. In the case of two broadly distributed rate processes crossing on an Arrhenius plot (i.e. the frequency-resolved, temperature-dependent dielectric loss spectrum of a and bh), it can be difficult to separate the two processes at a single temperature by assigning a fraction of the observed distribution to the two processes. Only by including a model of the temperature
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dependence of a and bh can a stable decomposition be obtained at their crossing, near 215 K [14]. Certainty that the processes cross comes only from the hydration and temperature dependence of bh, and the consistency of a’s temperature dependence between samples with and without protein (i.e. with and without bh). Unfortunately, many other papers do not provide enough information about their data evaluation procedure to support or reject this explanation of the dynamical crossover for their data. 6. Conclusions Neutron scattering and the Mössbauer effect yield significant data for protein dynamics, but a number of conceptual problems exist. Arguments, sometimes heated, have persisted for a long time. We hope that the present paper leads to reasoned discussion of the disputed questions so that the results from these techniques can be used with confidence. Acknowledgments We thank Joel Berendzen, Jason Lashley, and Dean Taylor for intense discussions that have been important for understanding the problems and difficulties in protein dynamics. References [1] S. Magazu, F. Migliardo, Dynamics of Biological Macromolecules by Neutron Scattering. Benthamscience eBook, 2011. [2] Y.-L. Chen, D.-P. Yang, Mössbauer Effect in Lattice Dynamics, Wiley-VCH, Weinheim, 2007. [3] R.M. May, PLoS Biol. 9 (2011) 8. [4] R.H. Austin, K.W. Beeson, L. Eisenstein, H. Frauenfelder, I.C. Gunsalus, Biochemistry 14 (1975) 5355. [5] H. Frauenfelder, F. Parak, R.D. Young, Annu. Rev. Biophys. Biophys. Chem. 17 (1988) 451. [6] A. Cooper, Prog. Biophys. Mol. Biol. 44 (1984) 181. [7] H. Frauenfelder, S.G. Sligar, P.G. Wolynes, Science 254 (1991) 1598. [8] F. Kremer, A. Schönhals, Broadband Dielectric Spectroscopy, Springer, 2003. [9] P.G. Wolynes, V. Lubchenko, Structural Glasses and Supercooled Liquids, Wiley, 2012. [10] K.L. Ngai, Relaxation and Diffusion in Complex Systems, Springer, 2011. [11] V. Lubchenko, P.G. Wolynes, H. Frauenfelder, J. Phys. Chem. B 109 (2005) 7488. [12] D.N. LeBard, D.V. Matyushov, J. Phys. Chem. B 114 (2010) 9246. [13] L. Zhang, L. Wang, Y.-T. Kao, W. Qiu, Y. Yang, O. Okobiah, D. Zhong, PNAS 104 (2007) 18461. [14] H. Frauenfelder, G. Chen, J. Berendzen, P.W. Fenimore, H. Jansson, B.H. McMahon, I.R. Stroe, J. Swenson, R.D. Young, Proc. Natl. Acad. Sci. 106 (2009) 5129. [15] J.G.P. Singh, F. Parak, S. Hunklinger, K. Dransfeld, Phys. Rev. Lett. 47 (1981) 685. [16] S.A. Lusceac, M.R. Vogel, C.R. Herbers, Biochim. Biophys. Acta 1804 (2010) 41. [17] L. Hong, N. Smolin, B. Lindner, A.P. Sokolov, J.C. Smith, Phys. Rev. Lett. 107 (2011) 148102. [18] D. Beece, L. Eisenstein, H. Frauenfelder, D. Good, M.C. Marden, L. Reinisch, A.H. Reynolds, L.B. Sorensen, K.T. Yue, Biochemisty 19 (1980) 5147. [19] T. Kleinert, W. Doster, H. Leyser, W. Petry, V. Schwarz, M. Settles, Biochemistry 37 (1998) 717. [20] H. Frauenfelder, P.W. Fenimore, B.H. McMahon, Biophys. Chem. 98 (2002) 35. [21] B.T.M. Willis, A.W. Pryor, Thermal Vibrations in Crystallography, Cambridge University Press, 1975. [22] H. Frauenfelder, G.A. Petsko, D. Tsernoglou, Nature 280 (1979) 558. [23] G.A. Petsko, D. Ringe, Ann. Rev. Biophys. Bioeng. 13 (1984) 331. [24] L. Maragliano, G. Cottone, L. Cordone, G. Ciccotti, Biophys. J. 86 (2004) 2765. [25] A.E. Garcia, J.A. Krumhansl, H. Frauenfelder, Proteins 29 (1997) 153. [26] H. Frauenfelder, R.D. Young, P.W. Fenimore, J. Phys. Chem. B, submitted for publication. [27] R.D. Young, H. Frauenfelder, P.W. Fenimore, Phys. Rev. Lett. 107 (2011) 158102. [28] F. Parak, H. Formanek, Acta Cryst. A 27 (1971) 573. [29] H. Keller, P.G. Debrunner, Phys. Rev. Lett. 45 (1980) 68. [30] S.G. Cohen, E.R. Bauminger, I. Nowik, S. Ofer, J. Yariv, Phys. Rev. Lett. 46 (1981) 1244. [31] F. Parak, E.N. Frolov, R.L. Mössbauer, V.I. Goldanskii, J. Mol. Biol. 145 (1981) 825. [32] W. Doster, S. Cusack, W. Petry, Nature 337 (1989) 754. [33] W. Doster, Biochim. Biophys. Acta 1804 (2010) 3. [34] W. Doster, S. Busch, A.M. Gaspar, M.-S. Appavou, J. Wuttke, H. Scheer, Phys. Rev. Lett. 104 (2010) 098101. [35] S. Magazu, F. Migliardo, A. Benedetto, J. Phys. Chem. B 115 (2011) 7736.
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Dr. Paul Fenimore received a B.S. from the Physics Department at Rensselaer Polytechnic Institute, and a Ph.D. from the Physics Department at the University of Illinois, Urbana-Champaign, He came to Los Alamos National Laboratory as a post-doc in 2001, and since 2004 he has been a staff scientist in the Theoretical Biology and Biophysics Group at Los Alamos National Laboratory. His published research includes papers on protein dynamics, function, and their coupling to solvent and hydration, mathematical epidemiology, and vaccine design. He is a recipient of two Los Alamos Small Team Distinguished Performance Awards.
Hans Frauenfelder received his Ph. D. in physics in 1950 from the Swiss Federal Institute of Technology. In 1952 he moved to the Physics Department of the University of Illinois. From 1992 to 2012 he was in the theory division of the Los Alamos National Laboratory. His research ranged from perturbed angular correlation to the Mössbauer effect and parity violation in nuclear physics. His current interest is protein dynamics.
Salvatore Magazù is Full Professor in Experimental Physics and Head of the research group in Structure of Matter and Biophysics at the Physics and Earth Sciences Department of Messina University, Italy. He is president of the Le Studium International Consortium COSMO (France) and of the Interuniversitary Consortium for Applied Physical Sciences CISFA (Italy); he has been associated researcher of Le Studium, Institute for Advanced Studies, at the laboratory Conditions Extrêmes et Matériaux : Haute Température et Irradiation CEMHTI – CNRS in Orléans; chairman and member of scientific committees at the European Synchrotron Radiation Facility and at the Institut Laue-Langevin in Grenoble. His research topics are manifold and, from some points of view, different, even if it emerges a common thread: the integrated employment of spectroscopic techniques, such as neutron scattering, synchrotron radiation spectroscopy and light scattering, addressed to characterize the structural and dynamical properties of systems of biophysical interest. He is author of more than 300 articles in international journals and more than 200 communications in conferences. He has received several international and national awards, such as the 2000 Scientia Europaea Prize by the French Academy of Sciences and Aventis.
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P.W. Fenimore et al. / Chemical Physics 424 (2013) 2–6 Ben McMahon is Deputy Group Leader in the Theoretical Biology and Biophysics Group at Los Alamos National Laboratory. His research interests range from the physical chemistry of protein dynamics and function to evolutionary analysis to systems-level modeling, such as epidemiology or disease progression.
Ferenc Mezei graduated with a Physics Diploma from Eötvös L. University, Budapest in 1965, and a Ph.D. from the Hungarian Academy of Sciences, Budapest in 1976. He has worked at Eötvös L. University, the Central Research Institute for Physics, Budapest, the InstitutLaue-Langevin, was professor at the Technical University, Berlin from 1984-2007, Director of the Berlin Neutron Scattering Center at the Hahn-Meitner-Institut, Berlin from 1992-1997 and 2001-2007, and at Los Alamos National Laboratory from 1997-2010, first as the John Wheatley Scholar and then as a Technical Staff Member from 2000-2010. He is an Ordinary Member of the Hungarian Academy of Sciences, a member of Academia Europaea, and former Chairman of the Action Committee on Publications of the European Physical Society 1989 – 1994. He has been awarded the Janossy Prize (1979), the Hewlett-Packard Europhysics Prize (1968), the Leo Szilard Medal (1996), the Middle Cross of the Republic of Hungary (1997), the Inaugural Walter Haelg Prize, and is a Fellow of the American Physical Society. He invented and developed neutron spin-echo and neutron supermirrors, provided experimental proof of the Landau-Khalatnikov relation for rotons in He, discovered the break-down of dynamic scaling in ferromagnets and dynamic scaling near glass transitions, and invented long pulse neutron sources, ballistic neutron guides, and multiplexing chopper system for pulsed spallation sources. He is currently the Head of the Target Division of the European Spallation Source and an adjunct professor at the University of California San Diego,.
Federica Migliardo is a biophysicist at the Department of Physics and Earth Sciences of the University of Messina, Italy. She has been ATER (Attaché Temporaire d’Enseignement et de Recherche) and UNESCO-L’Oréal International Fellow at the Laboratoire de Dynamique et Structure des Matériaux Moléculaires of the University of Lille 1 and European Molecular Biology Organisation (EMBO) International Fellow at the Institut de Biochimie et Biophysique Moléculaire et Cellulaire of the University of Paris-Sud XI, France. Her research activity is focused on the study of the molecular mechanisms responsible for biological processes, such as bioprotection, denaturation and stabilisation of biomolecules and, more recently, for infective (tuberculosis and schistosomiasis) and neurodegenerative (Parkinson) diseases. Her
methodological approach is based on the synergistic application of complementary techniques allowing to access to different space and time domains, in particular neutron and light scattering. For her research achievements, testified by more than 150 articles, she received several awards, such as, more recently, the International BioVision Award for Life Sciences 2011, the UNESCO-L’Oréal For Women in Science International Fellowship 2008 and the European EUWIIN Special Recognition Award 2007.
Robert D. Young is a physicist. He is currently adjunct faculty at Arizona State University. Prior to that, he was a professor and administrator at Illinois State University. He was also adjunct research professor at the University of Illinois at Champaign-Urbana. Early on, he specialized in elementary particle theory. In mid-career, biological physics became his consuming interest. He works on building dynamic models of proteins with environment included, based on fluctuations in their complex energy landscape. He currently resides in Flagstaff, Arizona, and enjoys being outside in the Grand Canyon of the Colorado River.
Izabela R. Stroe received her B.Sc. from the University of Bucharest in 1994 and her Ph.D. in Physics from Clark University in 2005. She joined the Los Alamos National Laboratory as a Seaborg Postdoctoral Fellow under the guidance of Dr. Albert Migliori working on the properties of actinides, correlated electrons systems and biological systems. In 2008 she joined the Physics Department at Worcester Polytechnic Institute as assistant professor tenure track. Her research aims at understanding the protein-solvent coupling using complementary experimental tools. She is a recipient of the Kalenian Award for innovation and entrepreneurship for her work on early detection of amyloidogenic precursors.