Dynamic Transition Associated with the Thermal Denaturation of a Small Beta Protein (original) (raw)
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New dynamic regimes in proteins revealed by neutron scattering
Understanding the mechanisms of protein folding requires knowledge of both the energy landscape and the structural dynamics of a protein. We report a neutron-scattering study of the nanosecond and picosecond dynamics of native and the denatured a-lactalbumin. The quasielastic scattering intensity shows that there are a-helical structure and tertiary- like side-chain interactions fluctuating on sub-nanosecond time-scales under extremely denaturing conditions and even in the absence of disul- fide bonds. Based on the length-scale dependence of the decay rate of the measured correlation functions, the nanosecond dynamics of the native and the variously denatured proteins have three dynamic regimes. When 0.05 < Q < 0.5 A˚ ÿ 1 (where the scattering vector, Q, is inversely pro- portional to the length-scale), the decay rate, ÿ, shows a power law relationship, ÿ / Q 2.42 0.08 , that is analogous to the dynamic behavior of a random coil. However, when 0.5 < Q < 1.0 A˚ ÿ 1 , the decay rate exhibits a ÿ / Q 1.0 0.2 relationship. The effective diffusion constant of the protein decreases with increasing Q, a striking dynamic behavior that is not found in any chain-like macromolecule. We suggest that this unusual dynamics is due to the presence of a strongly attractive force and collec- tive conformational fluctuations in both the native and the denatured states of the protein. Above Q > 1.0 A˚ ÿ 1 is a regime that displays the local dynamic behavior of individual residues, ÿ / Q 1.8 0.3 . The picose- cond time-scale dynamics shows that the potential barrier to side-chain proton jump motion is reduced in the molten globule and in the denatured proteins when compared to that of the native protein. Our results provide a dynamic view of the native-like topology established in the early stages of protein folding.
Dynamics of a globular protein as studied by neutron scattering and solid-state NMR
Physica B: Condensed Matter, 1997
The influence of hydration on the internal dynamics of a typical EF-hand calciprotein, parvalbumin, was investigated by incoherent quasi-elastic neutron scattering (IQNS) and solid-state 13 C-NMR spectroscopy using the powdered protein at different hydration levels. Both approaches establish an increase in protein dynamics upon progressive hydration above a threshold that only corresponds to partial coverage of the protein surface by the water molecules. Selective motions are apparent by NMR in the 10-ns time scale at the level of the polar lysyl side chains (externally located), as well as of more internally located side chains (from Ala and Ile), whereas IQNS monitors diffusive motions of hydrogen atoms in the protein at time scales up to 20 ps. Hydration-induced dynamics at the level of the abundant lysyl residues mainly involve the ammonium extremity of the side chain, as shown by NMR. The combined results suggest that peripheral water-protein interactions influence the protein dynamics in a global manner. There is a progressive induction of mobility at increasing hydration from the periphery toward the protein interior. This study gives a microscopic view of the structural and dynamic events following the hydration of a globular protein.
Dynamics of proteins at low temperatures: fibrous vs. globular
Applied Physics A: Materials Science & Processing, 2002
We have measured quasielastic neutron scattering from H 2 O-hydrated collagen and haemoglobin at T ≤ 270 K. The data consist of sets of nearly elastic peaks showing (i) Q,T -dependent decreases in window-integrated intensities S qe (Q;T ) proportional to effective Debye-Waller factors and (ii) small line-shape changes due to various types of proton motions with ns > τ > 10 ps. Relative to haemoglobin, the 200-K dynamic transition is shifted upward by 20-25 K in collagen, and the T -dependence of m.-sq. displacements derived from S qe (Q;T ) suggests that in triple-helical systems there are three rather than two regimes: one up to around 120 K (probably purely harmonic), an intermediate quasiharmonic region with a linear dependence up to ≈ 240 K, followed by a steeper nonlinear rise similar to that in globular proteins.
The Inverse Relationship between Protein Dynamics and Thermal Stability
Biophysical Journal, 2001
Protein powders that are dehydrated or mixed with a glassy compound are known to have improved thermal stability. We present elastic and quasielastic neutron scattering measurements of the global dynamics of lysozyme and ribonuclease A powders. In the absence of solvation water, both protein powders exhibit largely harmonic motions on the timescale of the measurements. Upon partial hydration, quasielastic scattering indicative of relaxational processes appears at sufficiently high temperature. When the scattering spectrum are analyzed with the Kohlrausch-Williams-Watts formalism, the exponent  decreases with increasing temperature, suggesting that multiple relaxation modes are emerging. When lysozyme was mixed with glycerol, its  values were higher than the hydrated sample at comparable temperatures, reflecting the viscosity and stabilizing effects of glycerol.
Internal motions in proteins: A combined neutron scattering and molecular modelling approach
Pramana, 2004
It is well-known that water plays a major role in the stability and catalytic function of proteins. Both the effect of hydration water on the dynamics of proteins and that of proteins on the dynamics of water have been studied using inelastic neutron scattering. Inelastic neutron scattering is the most direct probe of diffusive protein dynamics on the picosecond-nanosecond time-scale. We present here results relative to a photosynthetic globular protein, the C-phycocyanin, that can be obtained in protonated and deuterated forms. Diffusive motions have been studied using the protonated C-phycocyanin, protein. Molecular dynamics simulation and analytical theory have been combined to analyse the data and get a detailed description of diffusive motions for protein. The simulation-derived dynamic structure factors are in good agreement with experiment. The dynamical parameters are shown to present a smooth variation with distance from the core of the protein.
Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP
Biophysical Journal, 2012
We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (<~200 K), and facilitates protein dynamics at high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures <~180-190 K and is not affected by the proteins' methyl group rotations. The dynamics of the hydration water exhibits changes at~180-190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <~3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously observed for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equivalent hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.
Protein dynamics studied by neutron scattering
Quarterly Reviews of Biophysics, 2002
1. Introduction 3282. Basic concepts of neutron scattering 3292.1 Introduction 3292.2 Neutron-scattering functions 3312.3 Coherent and incoherent neutron scattering. The particular role of hydrogen in incoherent scattering 3322.4 Total elastic scattering, EISF and mean square displacement (MSD) 3332.5 Quasielastic scattering and relaxation function 3342.6 Inelastic scattering and density of states 3353. Experimental aspects and instruments 3353.1 Energy and space resolution 3353.2 General sample aspects 3353.3 Potential effects of D2O on dynamics 3363.4 Experimental 2H (deuterium) labelling 3364. Physics of protein dynamics 3364.1 Models 3364.2 The dynamical transition 3384.3 Effective force constants 3395. Dynamics of hydrated protein powders 3395.1 First experiments on myoglobin 3405.2 Dynamical transitions in other proteins 3405.3 The role of hydration water 3415.4 Influence of the solvent 3445.5 Diffusional motions within proteins by QENS 3465.6 Inelastic neutron scattering and ...
Spectroscopy, 2010
This review article describes our neutron scattering experiments made in the past four years for the understanding of the single-particle (hydrogen atom) dynamics of a protein and its hydration water and the strong coupling between them. We found that the key to this strong coupling is the existence of a fragile-to-strong dynamic crossover (FSC) phenomenon occurring at around T L = 225 ± 5 K in the hydration water. On lowering of the temperature toward FSC, the structure of hydration water makes a transition from predominantly the high density form (HDL), a more fluid state, to predominantly the low density form (LDL), a less fluid state, derived from the existence of a liquid-liquid critical point at an elevated pressure. We show experimentally that this sudden switch in the mobility of hydration water on Lysozyme, B-DNA and RNA triggers the dynamic transition, at a temperature T D = 220 K, for these biopolymers. In the glassy state, below T D , the biopolymers lose their vital conformational flexibility resulting in a substantial diminishing of their biological functions. We also performed molecular dynamics (MD) simulations on a realistic model of hydrated lysozyme powder, which confirms the existence of the FSC and the hydration level dependence of the FSC temperature. Furthermore, we show a striking feature in the short time relaxation (β-relaxation) of protein dynamics, which is the logarithmic decay spanning 3 decades (from ps to ns). The long time α-relaxation shows instead a diffusive behavior, which supports the liquid-like motions of protein constituents. We then discuss our recent high-resolution X-ray inelastic scattering studies of globular proteins, Lysozyme and Bovine Serum Albumin. We were able to measure the dispersion relations of collective, intra-protein phonon-like excitations in these proteins for the first time. We found that the phonon energies show a marked softening and at the same time their population increases substantially in a certain wave vector range when temperature crosses over the T D . Thus the increase of biological activities above T D has positive correlation with activation of slower and large amplitude collective motions of a protein.
Journal of Molecular Biology, 2001
Neocarzinostatin is an all-b protein, 113 amino acid residues long, with an immunoglobulin-like fold. Its thermal unfolding has been studied by small-angle X-ray scattering. Preliminary differential scanning calorimetry and¯uorescence measurements suggest that the transition is not a simple, two-state transition. The apparent radius of gyration is determined using three different approaches, the validity of which is critically assessed using our experimental data as well as a simple, two-state model. Similarly, each step of data analysis is evaluated and the underlying assumptions plainly stated. The existence of at least one intermediate state is formally demonstrated by a singular value decomposition of the set of scattering patterns. We assume that the pattern of the solution before the onset of the transition is that of the native protein, and that of the solution at the highest temperature is that of the completely unfolded protein. Given these, actually not very restrictive, boundary constraints, a least-squares procedure yields a scattering pattern of the intermediate state. However, this solution is not unique: a whole class of possible solutions is derived by adding to the previous linear combination of the native and completely unfolded states. Varying the initial conditions of the least-squares calculation leads to very similar solutions. Whatever member of the class is considered, the conformation of this intermediate state appears to be weakly structured, probably less than the transition state should be according to some proposals. Finally, we tried and used the classical model of three thermodynamically well-de®ned states to account for our data. The failure of the simple thermodynamic model suggests that there is more than the single intermediate structure required by singular value decomposition analysis. Formally, there could be several discrete intermediate species at equilibrium, or an ensemble of conformations differently populated according to the temperature. In the latter case, a third state would be a weighted average of all non native and not completely unfolded states of the protein but, since the weights change with temperature, no meaningful curve is likely to be derived by a global analysis using the simple model of three thermodynamically well-de®ned states.
Journal of Molecular Biology, 2001
Neocarzinostatin is an all-b protein, 113 amino acid residues long, with an immunoglobulin-like fold. Its thermal unfolding has been studied by small-angle X-ray scattering. Preliminary differential scanning calorimetry and¯uorescence measurements suggest that the transition is not a simple, two-state transition. The apparent radius of gyration is determined using three different approaches, the validity of which is critically assessed using our experimental data as well as a simple, two-state model. Similarly, each step of data analysis is evaluated and the underlying assumptions plainly stated. The existence of at least one intermediate state is formally demonstrated by a singular value decomposition of the set of scattering patterns. We assume that the pattern of the solution before the onset of the transition is that of the native protein, and that of the solution at the highest temperature is that of the completely unfolded protein. Given these, actually not very restrictive, boundary constraints, a least-squares procedure yields a scattering pattern of the intermediate state. However, this solution is not unique: a whole class of possible solutions is derived by adding to the previous linear combination of the native and completely unfolded states. Varying the initial conditions of the least-squares calculation leads to very similar solutions. Whatever member of the class is considered, the conformation of this intermediate state appears to be weakly structured, probably less than the transition state should be according to some proposals. Finally, we tried and used the classical model of three thermodynamically well-de®ned states to account for our data. The failure of the simple thermodynamic model suggests that there is more than the single intermediate structure required by singular value decomposition analysis. Formally, there could be several discrete intermediate species at equilibrium, or an ensemble of conformations differently populated according to the temperature. In the latter case, a third state would be a weighted average of all non native and not completely unfolded states of the protein but, since the weights change with temperature, no meaningful curve is likely to be derived by a global analysis using the simple model of three thermodynamically well-de®ned states.