Direct Experimental Characterization of Contributions from Self-Motion

Subscriber access provided by UNIV OF WATERLOO

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Direct Experimental Characterization of Contributions from Self-Motion of Hydrogen and from Interatomic Motion of Heavy Atoms to Protein Anharmonicity Zhuo Liu, Chenxing Yang, Juan Huang, Gaia Ciampalini, Jun Li, Victoria García-Sakai, Madhusudan Tyagi, Hugh M. O'Neill, Qiu Zhang, Simone Capaccioli, Kia L. Ngai, and Liang Hong J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Direct Experimental Characterization of Contributions from Self-motion of Hydrogen and from Interatomic Motion of Heavy Atoms to Protein Anharmonicity †

†

‡

§

†

Zhuo Liu, ,¶ Chenxing Yang, ,¶ Juan Huang, Gaia Ciampalini, Jun Li, Victoria García Sakai, ‖

Madhusudan Tyagi,⊥ Hugh O’Neill,∥ Qiu Zhang,∥ Simone Capaccioli,§ Kia Ling Ngai,§ and

Liang Hong*,†,∆ †

School of Physics and Astronomy, Shanghai Jiao Tong University, Shanghai 200240, P. R.

China ‡

School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240,

P. R. China §

Dipartimento di Fisica “E. Fermi”, Università di Pisa and Istituto peri Processi Chimico-

Fisici—Consiglio Nazionale delle Ricerche, Largo Pontecorvo 3, Pisa 56127, Italy ‖

ISIS Neutron and Muon Facility, Rutherford Appleton Laboratory, Science & Technology

Facilities Council, Didcot, OX11 0QX, United Kingdom

ACS Paragon Plus Environment

1

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

⊥

Page 2 of 18

NIST Center for Neutron Research, National Institute of Standards and Technology (NIST),

Gaithersburg, MD 20899, United States & Department of Materials Science and Engineering, University of Maryland, College Park, MD 20742, United States ∥

Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37931,

United States ∆

Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai 200240, P. R. China

*Corresponding Author E-mail: [email protected]


2

Page 3 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Abstract

One fundamental challenge in biophysics is to understand the connection between protein dynamics and its function. Part of the difficulty arises from the fact that proteins often present local atomic motions and collective dynamics on the same time scales, and challenge the experimental identification and quantification of different dynamic modes. Here, by taking lyophilized proteins as example, we combined deuteration technique and neutron scattering to separate and characterize the self-motion of hydrogen and the collective interatomic motion of heavy atoms (C, O, N) in proteins on the pico-to-nanosecond time scales. We found that hydrogen atoms present an instrument-resolution-dependent onset for anharmonic motions, which can be ascribed to the thermal activation of local side-group motions. However, the protein heavy atoms exhibit an instrument-resolution-independent anharmonicity around 200 K, which results from unfreezing of the relaxation of the protein structures on the laboratory equilibrium time (100-1000 s), softening of the entire bio-macromolecules.


3


Page 4 of 18

Introduction Protein internal motions are intimately connected to its enzymatic function and folding.1-4 It has been widely discovered that the pico-to-nanosecond dynamics in hydrated proteins will present a harmonic to anharmonic transition around ~200 K, where the slope of the temperature dependence of the mean-squared atomic displacement of protein atoms presents a rapid increase, and this transition has been connected to the onset of protein function.3, 5-6 Moreover, the meansquared atomic displacement on these time scales in various freeze-dried proteins is found to linearly correlate with the degradation rate of the enzymes, i.e., higher flexibility corresponds to faster degradation.7-8 However, on the pico-to-nanosecond time scales, protein dynamics consists of both local side-group motions, such as methyl rotation and flipping of benzene rings, and collective interatomic motions involving the protein backbone.1, 9-13 Experimental exploration of the spatial-temporal features and temperature dependence of these distinct dynamical modes could be important towards understanding the connection between the protein internal motions and its function and stability. Such work is, however, largely lacking due to the experimental difficulty to explicitly separate the different atomic motions on the same time scales. The above difficulty can be overcome by neutron scattering on proteins with proper hydrogen/deuterium contrast. As shown in Ref. 9, the neutron signals collected on hydrogenated proteins is mostly incoherent (~90%), resulting from self-motions of protein hydrogen atoms, while the neutron scattering on perdeuterated samples is primarily coherent (~90%), arising mostly from interatomic motions of protein heavy atoms (C, N, O), which have been demonstrated highly correlated over a few nanometers.10 Herein, by performing neutron scattering on both cytochrome P450 101 (CYP101, Figure 1a) and green fluorescent protein (GFP, Figure 1b) and their perdeuterated counterparts, the contributions of the self-motion of


4

Page 5 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydrogen atoms and the interatomic motion of heavy atoms to the protein anharmonicity on the pico-to-nanosecond time scales are characterized separately. It is found that the hydrogen atoms present an instrument-resolution-time dependent anharmonic onset, while the interatomic motion of heavy atoms exhibit a resolution-independent anharmonicity around 200K. Further analyses of the experimental data and the Molecular Dynamics (MD) simulation trajectories reveal that the anharmonicity of hydrogen atoms results primarily from the thermal activation of methyl rotations, while that of the protein heavy atoms comes from unfreezing of the relaxation of the protein structures within the experimental equilibrium time (100-1000 s), which renders global flexibility to the bio-macromolecules. Results and Discussion Deuteration is key to the present work. For simplicity, in what follows hydrogenated and perdeuterated samples are denoted using the prefixes of H and D. To suppress the contribution of water to the neutron signals, all the protein samples studied were lyophilized for about 12 hours, where the residue water is ~2% in weight as determined by the Thermogravimetric Analyzer (see Figure S1 in the Supporting Information, SI). Herein, D-CYP101 and D-GFP and their Hcounterparts were examined by three neutron instruments: HFBS, OSIRIS and IRIS, with distinct energy resolutions, ∆E, of 1 µeV, 25.4 µeV and 17.5 µeV, respectively. These energy resolutions correspond to different time resolutions, ∆t, of 1 ns, 40 ps and 60 ps, respectively. The experimentally measured quantity is the intensity of the elastic peak of the dynamic structure factor, S(q, ∆t), which provides an estimate of the average amplitude of the atomic motions occurring at times up to the instrumental resolution time, ∆t. More details about the experimental technique, sample preparation, data analysis and MD simulations were presented in SI.


5


Page 6 of 18

Figure 1 presents the temperature dependence of S(q, ∆t) for both lyophilized CYP101 and GFP. The results for H-protein were plotted in Figures 1c and 1e, exhibiting drastic differences when examined by instruments of different resolution times. Take H-CYP101 for example (Figure 1c), when measured using HFBS (∆t = 1 ns), S(q, ∆t) bends downwards upon heating around 150 K, indicating an onset for anharmonic atomic motions at the corresponding temperature. More importantly, the onset temperature shifts to ~200 K when reducing the instrument resolution time to 40 ps by using OSIRIS. These results are further confirmed by Figure 2a when analyzing the corresponding mean-squared atomic displacement, MSD (detailed procedures to derive MSD can be found in SI).


6

Page 7 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Structures of (a) CYP101 and (b) GFP. Experimental S(q, ∆t), normalized to the lowest temperatures (∼10 K) and summed over the q range from 0.45 to 1.75 Å−1 for lyophilized (c) HCYP101, (d) D-CYP101, and (e) H-GFP at the resolution times of 1 ns (black) and 40 ps (red), and for lyophilized (f) D-GFP at the resolution times of 1 ns (black) and 60 ps (red). Normalizations to the Vanadium value instead of the lowest-temperature value will not change the results qualitatively.9 For clarity, the experimental results of S(q, ∆t) at 1 ns (black) and 40/60 ps (red) are grouped in intervals of 6 K and 15 K, respectively. For quantitative analysis, the onset temperature, Ton, is defined as the crossing point of two linear fits (dashed lines) in the high (220 to 300 K) and low (5-100K) temperature regions.9 The same procedure is applied in Figure 2 to determine Ton from the mean-squared atomic displacements. The elastic-scan data of lyophilized D-proteins at 1 ns were reported in Ref. 9 to demonstrate that the 200 K anharmonic onset is present in lyophilized proteins.

The observed dependence of Ton on ∆t is a typical behavior for a thermally-activated barrierhopping process, e.g., Arrhenius type process, to enter the time window of the instrument when its characteristic relaxation time becomes comparable to the instrumental resolution, ∆t.14 Here, by assuming an Arrhenius-type barrier-hopping process, τ=τ0*exp(∆U/kBT), this set of data (Ton vs. ∆t) yields an energy barrier, ∆U ~3.3 kcal/mol for H-CYP101. Similar results were obtained when analyzing S(q, ∆t) data of H-GFP (Figure 1e), for which ∆U is estimated to be ~2.8 kcal/mol. The obtained ∆U is in quantitative agreement with the energy barriers reported previously for methyl rotation in proteins,15-17 which was known to obey Arrhenius-type dependence.14,

18

These results are also consistent with the findings from the early neutron


7


Page 8 of 18

scattering experiments performed on homomeric polypeptides that only the methyl-contained homopolymer presents the anharmonic onset at ~150 K when ∆t is 1 ns.14

Figure 2. Experimental mean-squared atomic displacements derived from lyophilized (a) H-CYP101, (b) D-CYP101, (c) H-GFP, and (d) D-GFP. Detailed procedures to derive are presented in the Supporting Information. Error bars throughout the text represent one standard deviation.

To further understand the anharmonic onset found in lyophilized H-proteins, we performed all-atom MD simulations at the corresponding experimental conditions. As shown by Figure S2 in SI, the simulations explicitly demonstrated that it is the methyl rotation entering the instrumental time window that produces the resolution-dependent anharmonic onset observed in H-CYP101. The instrument-resolution dependence of the MD-derived Ton resulting from methyl rotations (Figure S2) is qualitatively similar as that found in experiment (Figure 1c). Therefore,


8

Page 9 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


we attribute the anharmonic onset discovered in lyophilized H-proteins primarily to the thermal activation of methyl rotations with an activation barrier of ~3 kcal/mol, which enter the experimental time window at Ton. The S(q, ∆t) measured on D-proteins is plotted in Figures 1d and 1f, which exhibits drastic differences as compared to that of H-proteins (Figures 1c and 1e). First of all, it shows much weaker temperature dependence, and this can result from the fact that the neutron signals from D-proteins are primarily coherent, reflecting interatomic motions of heavy atoms,9,

14

having

much smaller atomic displacement than that of hydrogen. Secondly and more importantly, both the anharmonic onset temperature (~200 K) and the temperature variations of the dynamics [S(q, ∆t) and MSD] throughout the entire temperature range from 10 K to 300K for D-proteins present little dependence on the instrumental resolution (Figures 1d, 1f, 2b and 2d). Here, the value of “200 K” is only a rough estimate for Ton of D-proteins, and the exact value can vary slightly with the type of proteins and also with the presentation of neutron data as S(q, ∆t) or MSD (Figures 1 and 2). As discovered by Ref. 19, hydrated homomeric polypeptides also present a resolutionindependent anharmonic onset around 200 K, which has been attributed to low-density to highdensity liquid-liquid transition. The anharmonic onset identified in D-proteins is unlikely resulting from side-group motions. Firstly, side-group motions are often barrier-hopping process with certain energy barrier, and the activation temperature for the process to enter the experimental time window will significantly change with the instrumental resolution (e.g., see Figures 1c, 1e, 2a and 2c). Secondly, neutron signals collected on perdeuterated protein are mostly coherent signals, reflecting the interatomic motion of heavy atoms, especially the backbone heavy atoms, which have been demonstrated highly collective over a few nanometers on the pico-to-nanosecond time scales.10 In contrast,


9


Page 10 of 18

side group motions, e.g., methyl rotations, are normally considered to be localized, independent atomic motions.13-14 Thirdly, as shown in Figure 3a, the mechanical modulus of lyophilized GFP presents a rapid drop around 200 K upon heating., i.e., softening of the entire protein molecules.9 Thermal activation of local side-group motion alone is unlikely to induce significant change of the global mechanical properties of the bio-macromolecules. As the neutron scattering measurement at each temperature will take several minutes, a plausible mechanism for the ~200 K onset discovered in D-protein could be that the relaxation of protein structure is slowed down upon cooling and eventually exits out the experimental equilibrium time (100-1000 s), i.e., freezing of the structural relaxation on the laboratory time below 200 K. As demonstrated for various soft-matter systems ranging from small molecular systems to polymers, when the characteristic time of either the α relaxation or the JohariGoldstein (βJG) relaxation falls out of the experimental equilibrium time upon cooling, the entire system will be mechanically rigidified with the fast pico-to-nanosecond atomic fluctuation being suppresed.20-24 To verify the above hypothesis, we carried out the measurement of the dielectric loss as a function of frequencies in the lyophilized H-CYP101 at various temperatures by using a Novocontrol dielectric analyzer. We used lyophilized H-protein instead of D-protein as the Dprotein was very expensive. However, the dielectric susceptibility data we obtained from the Hprotein should be the same as that from the D-protein. As seen in Figure 3b, a pronounced peak in dielectric loss is present in the broad frequency range from 10-2 to 105 Hz, indicating that a single relaxation process involving fluctuation of dipole moments is dominant in the corresponding time window. Such relaxation process was also reported for other dry proteins and has been assigned as relaxation of protein structures involving polar groups.25-26


10

Page 11 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (a) Longitudinal modulus measured using Brillouin light scattering on lyophilized HGFP (the data were taken from Ref. 9). Blue arrow in (a) marks the kink in the temperature dependence of the modulus. (b) Dielectric loss spectra of lyophilized H-CYP101 measured at selective temperatures. (c) Relaxation times of H-CYP101 derived from (b) as a function of reciprocal temperature. The red dashed line is a linear extrapolation towards the low temperatures, and the blue shaded area indicates the time window from 100 to 1000 s. To obtain a complete dielectric spectrum with a well-resolved loss peak, one needs to perform the experiment for a time span, which is at least two or three orders of magnitude longer than the characteristic time at the peak. Therefore, to directly measure the spectra with a peak at 1001000 s, one needs to perform measurement on one sample for days, which not only takes too much time but might also trigger some instability during the measurement. That is the reason we applied the extrapolation from the short-time data at high temperature, which is widely used in


11


Page 12 of 18

literature to determine the characteristic temperature of soft matter systems for the relaxation time on 100-1000 sec.27-30

A characteristic relaxation time (τ) can be derived from the peak position in the dielectric loss spectrum, and the resulting τ is plotted as a function of temperature in Figure 3c. The extrapolation of the temperature dependence of τ to the region of 100 to 1000 s roughly crosses at ~200 K. Hence, the relaxation of protein structures is frozen out of the laboratory equilibrium time when cooling below 200 K. Consequently, this will stiffen the entire materials (Figure 3a) as normally observed in soft-matter systems.21-24 Such global rigidification can strongly suppress the fast pico-to-nanosecond fluctuation of the protein backbone, which contributes significantly to the coherent neutron signals collected on D-proteins,9 which mainly reflects the collective inter-heavy-atom motions. In contrast, such variation of global mechanical properties is unlikely to strongly affect the local side-group motions, e.g., methyl rotations, which dominate the neutron signals measured on lyophilized H-proteins. Furthermore, this provides a rational explanation for why the anharmonic onset discovered in D-proteins is resolution-timeindependent. This is because freezing of the relaxation of the protein structure should occur around one fixed temperature when its characteristic relaxation time reaches the laboratory equilibrium time (100-1000 s, Figure 3c), and thus it will not depend on whether the resolution of the neutron instrument is set as 1 ns or 40 ps.

Conclusion


12

Page 13 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


By means of neutron scattering measurements on lyophilized hydrogenated and perdeuterated proteins using instruments of different resolutions, the self-motion of hydrogen atoms and the collective interatomic motion of heavy atoms in proteins are characterized separately over a wide temperature range and at distinct time scales. The self-motion of hydrogen atoms presents a resolution-time-dependent anharmonic onset. It can be attributed to the thermal activation of local side-group motions, mostly methyl rotation, following an Arrhenius law with an energy barrier of ~3 kcal/mol. In contrast, the collective interatomic motion of heavy atoms exhibits a resolution-independent onset around 200 K, which comes from unfreezing of the relaxation of protein structures beyond the laboratory equilibrium time (100-1000 s) upon heating.

The resolution-independent anharmonicity of heavy-atom motions discovered here could be an intrinsic nature of lyophilized proteins, independent on the structures of the bio macromolecules as the two proteins studied here differ significantly in both secondary and tertiary structures (Figures 1a and 1b). Whether the 200 K anharmonic onset discovered here in lyophilized protein has the same physical origin as the one found in hydrated protein powder is unclear. However, this finding on lyophilized proteins could be important by itself. Firstly, as reported by Ref. 31-32, both Candida rugosa Lipase B and pig liver esterase at a very dry condition (≤ 5% water) can still perform catalytic functions as long as the substrate are fed in gas phase. The anharmonic onset discovered here, i.e., the released degree of freedom for the collective heavy-atom motions above 200K, might provide the dynamical origin for the observed residual bioactivity in these dry enzymes. Moreover, due to the marginal stability of proteins in the aqueous state, pharmaceutic protein products are often preserved in the lyophilized solid state to ensure the long-term stability.33-34 However, even preserved in lyophilized state, protein molecules can still undergo anharmonic motions to denature. The anharmonic onset discovered here furnishes the


13


Page 14 of 18

global mechanical softness to the lyophilized protein and unfreezes the relaxation of its structures, both of which might facilitate the large-scale long-time conformational motions of the protein molecule and the diffusion of denature agents inside the preserved protein products.7 All of these could be of direct relevance to stability of the bio macromolecules stored in the freezedried solid state, which deserves further investigation.

AUTHOR INFORMATION Corresponding Author *(L. H.) E-mail: [email protected] Author Contributions ¶

These authors contributed equally.

Notes The authors declare no competing financial interests.

Supporting Information

Details of the sample preparation, neutron scattering experiments, dielectric spectroscopy measurements and MD simulations.

ACKNOWLEDGMENT This work was supported by NSF China 11504231, 31630002, and the Innovation Program of Shanghai Municipal Education Commission. H.O’N and Q.Z. acknowledge the support of Center


14

Page 15 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for Structural Molecular Biology (FWP ERKP291) funded by the U. S. Department of Energy (DOE) Office of Biological and Environmental Research. Z. L. acknowledges the Visiting Student Program of University of Pisa supporting the stay at Pisa in Italy.

REFERENCES (1) Henzler-Wildman, K.; Kern, D. Dynamic personalities of proteins. Nature 2007, 450, 964. (2) Snow, C. D.; Nguyen, H.; Pande, V. S.; Gruebele, M. Absolute comparison of simulated and experimental protein-folding dynamics. Nature 2002, 420, 102. (3) Rasmussen, B. F.; Stock, A. M.; Ringe, D.; Petsko, G. A. Crystalline ribonuclease A loses function below the dynamical transition at 220 K. Nature 1992, 357, 423-424. (4) Frauenfelder, H.; Sligar, S. G.; Wolynes, P. G. The energy landscapes and motions of proteins. Science 1991, 254, 1598. (5) R.M. Daniel; R.V. Dunn; J.L. Finney; Smith, J. C. The Role of Dynamics in Enzyme Activity. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 69-92. (6) Rupley, J. A.; Careri, G. Protein Hydration and Function. Adv. Protein Chem. 1991, 41, 37-172. (7) Cicerone, M.; Douglas, J. β-Relaxation governs protein stability in sugar-glass matrices. Soft Matter 2012, 8, 2983-2991. (8) Wang, B.; Tchessalov, S.; Cicerone, M. T.; Warne, N. W.; Pikal, M. J. Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility. J. Pharm. Sci. 2009, 98, 3145-3166. (9) Liu, Z.; Huang, J.; Tyagi, M.; O’Neill, H.; Zhang, Q.; Mamontov, E.; Jain, N.; Wang, Y.; Zhang, J.; Smith, J. C., et al. Dynamical Transition of Collective Motions in Dry Proteins. Phys. Rev. Lett. 2017, 119, 048101. (10) Hong, L.; Jain, N.; Cheng, X.; Bernal, A.; Tyagi, M.; Smith, J. C. Determination of functional collective motions in a protein at atomic resolution using coherent neutron scattering. Sci. Adv. 2016, 2, e1600886. (11) Nickels, Jonathan D.; Perticaroli, S.; O’Neill, H.; Zhang, Q.; Ehlers, G.; Sokolov, Alexei P. Coherent Neutron Scattering and Collective Dynamics in the Protein, GFP. Biophys. J . 2013, 105, 2182-2187. (12) Lee, A. L.; Wand, A. J. Microscopic origins of entropy, heat capacity and the glass transition in proteins. Nature 2001, 411, 501. (13) Lewandowski, J. R.; Halse, M. E.; Blackledge, M.; Emsley, L. Direct observation of hierarchical protein dynamics. Science 2015, 348, 578. (14) Schiró, G.; Caronna, C.; Natali, F.; Cupane, A. Direct Evidence of the Amino Acid Side Chain and Backbone Contributions to Protein Anharmonicity. J. Am. Chem. Soc. 2010, 132, 1371-1376. (15) Xue, Y.; Pavlova, M. S.; Ryabov, Y. E.; Reif, B.; Skrynnikov, N. R. Methyl rotation barriers in proteins from 2H relaxation data. Implications for protein structure. J. Am. Chem. Soc. 2007, 129, 6827-38.


15


Page 16 of 18

(16) Hong, L.; Smolin, N.; Lindner, B.; Sokolov, A. P.; Smith, J. C. Three Classes of Motion in the Dynamic Neutron-Scattering Susceptibility of a Globular Protein. Phys. Rev. Lett. 2011, 107, 148102. (17) Roh, J.; Novikov, V.; Gregory, R.; Curtis, J.; Chowdhuri, Z.; P Sokolov, A. Onsets of Anharmonicity in Protein Dynamics. Phys. Rev. Lett. 2005, 95, 038101. (18) Frick, B.; Fetters, L. J. Methyl Group Dynamics in Glassy Polyisoprene: A Neutron Backscattering Investigation. Macromolecules 1994, 27, 974-980. (19) Schiró, G.; Natali, F.; Cupane, A. Physical Origin of Anharmonic Dynamics in Proteins: New Insights From Resolution-Dependent Neutron Scattering on Homomeric Polypeptides. Phys. Rev. Lett. 2012, 109, 128102. (20) Angell, C. A. Ten questions on glassformers, and a real space `excitations' model with some answers on fragility and phase transitions. J. Phys.: Condens. Matter 2000, 12, 6463. (21) Ngai, K. L.; Capaccioli, S.; Prevosto, D.; Wang, L.-M. Coupling of Caged Molecule Dynamics to JG β-Relaxation III:van der Waals Glasses. J. Phys. Chem. B 2015, 119, 1251912525. (22) Ngai, K. L.; Capaccioli, S.; Prevosto, D.; Wang, L.-M. Coupling of Caged Molecule Dynamics to JG β-Relaxation II: Polymers. J. Phys. Chem. B 2015, 119, 12502-12518. (23) Terki, F.; Levelut, C.; Prat, J. L.; Boissier, M.; Pelous, J. Low-frequency dynamics in an optical strong glass: vibrational and relaxational contributions. J. Phys.: Condens. Matter 1997, 9, 3955. (24) Forrest, J. A.; Dalnoki-Veress, K.; Dutcher, J. R. Interface and chain confinement effects on the glass transition temperature of thin polymer films. Phys. Rev. E 1997, 56, 5705-5716. (25) Khodadadi, S.; Pawlus, S.; Sokolov, A. P. Influence of Hydration on Protein Dynamics: Combining Dielectric and Neutron Scattering Spectroscopy Data. J. Phys. Chem. B 2008, 112, 14273-14280. (26) Panagopoulou, A.; Kyritsis, A.; Shinyashiki, N.; Pissis, P. Protein and Water Dynamics in Bovine Serum Albumin–Water Mixtures over Wide Ranges of Composition. J. Phys. Chem. B 2012, 116, 4593-4602. (27) Trachenko, K.; Roland, C. M.; Casalini, R. Relationship between the Nonexponentiality of Relaxation and Relaxation Time in the Problem of Glass Transition. J. Phys. Chem. B 2008, 112, 5111-5115. (28) Fomina, M.; Schirò, G.; Cupane, A. Hydration dependence of myoglobin dynamics studied with elastic neutron scattering, differential scanning calorimetry and broadband dielectric spectroscopy. Biophys. Chem. 2014, 185, 25-31. (29) Jansson, H.; Bergman, R.; Swenson, J. Relation between Solvent and Protein Dynamics as Studied by Dielectric Spectroscopy. J. Phys. Chem. B 2005, 109, 24134-24141. (30) Shinyashiki, N.; Yamamoto, W.; Yokoyama, A.; Yoshinari, T.; Yagihara, S.; Kita, R.; Ngai, K. L.; Capaccioli, S. Glass Transitions in Aqueous Solutions of Protein (Bovine Serum Albumin). J. Phys. Chem. B 2009, 113, 14448-14456. (31) Lind, P. A.; Daniel, R. M.; Monk, C.; Dunn, R. V. Esterase catalysis of substrate vapour: enzyme activity occurs at very low hydration. Biochim. Biophys. Acta, Proteins Proteomics 2004, 1702, 103-110. (32) Lopez, M.; Kurkal-Siebert, V.; Dunn, R. V.; Tehei, M.; Finney, J. L.; Smith, Jeremy C.; Daniel, R. M. Activity and Dynamics of an Enzyme, Pig Liver Esterase, in Near-Anhydrous Conditions. Biophys. J . 2010, 99, L62-L64.


16

Page 17 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(33) Carpenter, J. F.; Manning, M. C.; Randolph, T. W. Long-Term Storage of Proteins. Curr. Protoc. Protein Sci. 2002, 27, 4.6.1-4.6.6. (34) Simpson, R. J. Stabilization of Proteins for Storage. Cold Spring Harb. Protoc. 2010, 2010, doi:10.1101/pdb.top79.


17


Page 18 of 18

TOC Graphic


18

Direct Experimental Characterization of Contributions from Self-Motion

Recommend Documents