The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

How a vicinal layer of solvent modulates the dynamics of proteins

The dynamics of a folded protein is studied in water and glycerol at a series of temperatures below and above their respective dynamical transition. The system is modeled in two distinct states whereby the protein is decoupled from the bulk solvent at low temperatures, and communicates with it through a vicinal layer at physiological temperatures. A linear viscoelastic model elucidates the less-than-expected increase in the relaxation times observed in the backbone dynamics of the protein. The model further explains the increase in the flexibility of the protein once the transition takes place and the differences in the flexibility under the different solvent environments. Coupling between the vicinal layer and the protein fluctuations is necessary to interpret these observations. The vicinal layer is postulated to form once a threshold for the volumetric fluctuations in the protein to accommodate solvents of different sizes is reached. Compensation of entropic-energetic contributions from the protein-coupled vicinal layer quantifies the scaling of the dynamical transition temperatures in various solvents. The protein adapts different conformational routes for organizing the required coupling to a specific solvent, which is achieved by adjusting the amount of conformational jumps in the surface-group dihedrals.     

The protein-solvent glass transition

The protein dynamical transition and its connection with the liquid-glass transition (GT) of hydration water and aqueous solvents are reviewed. The protein solvation shell exhibits a regular glass transition, characterized by steps in the specific heat and the thermal expansion coefficient at the calorimetric glass temperature T_G ≈ 170 K. It implies that the time scale of the structural α-relaxation has reached the experimental time window of 1–100 s. The protein dynamical transition, identified from elastic neutron scattering experiments by enhanced amplitudes of molecular motions exceeding the vibrational level [1], probes the α-process on a shorter time scale. The corresponding liquid-glass transition occurs at higher temperatures, typically 240 K. The GT is generally associated with diverging viscosities, the freezing of long-range translational diffusion in the supercooled liquid. Due to mutual hydrogen bonding, both, protein- and solvent relaxational degrees of freedom slow down in paralled near the GT. However, the freezing of protein motions, where surface-coupled rotational and librational degrees of freedom are arrested, is better characterized as a rubber-glass transition. In contrast, internal protein modes such as the rotation of side chains are not affected. Moreover, ligand binding experiments with myoglobin in various glass-forming solvents show, that only ligand entry and exit rates depend on the local viscosity near the protein surface, but protein-internal ligand migration is not coupled to the solvent. The GT leads to structural arrest on a macroscopic scale due to the microscopic cage effect on the scale of the intermolecular distance. Mode coupling theory provides a theoretical framework to understand the microcopic nature of the GT even in complex systems. The role of the α- and β-process in the dynamics of protein hydration water is evaluated. The protein-solvent GT is triggered by hydrogen bond fluctuations, which give rise to fast β-processes. High-frequency neutron scattering spectra indicate increasing hydrogen bond braking above T_G.     

Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals

Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.     

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca²⁺ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca²⁺ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein’s dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.     

Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad

The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/β-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/β-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.     

The role of protein–solvent hydrogen bond dynamics in the structural relaxation of a protein in glycerol versus water

We used MD simulations to investigate the dependence of the dynamics of a soluble protein, RNase A, on temperature and solvent environment. Consistent with neutron scattering data, the simulations predict that the protein undergoes a dynamical transition in both glycerol and aqueous solutions that is absent in the dry protein. The temperature of the transition is higher, while the rate of increase with temperature of the amplitudes of motion on the 100 ps timescale is lower, in glycerol versus water. Analysis of the dynamics of hydrogen bonds revealed that the protein dynamical transition is connected to the relaxation of the protein-solvent hydrogen bond network, which, in turn, is associated with solvent translational diffusion. Thus, it appears that the role of solvent dynamics in affecting the protein dynamical transition is qualitatively similar in water and glycerol.     

The 'glass transition' in protein dynamics: what it is, why it occurs, and how to exploit it

All proteins undergo a dramatic change in their dynamical properties at approximately 200 K. Above this temperature, their dynamic behavior is dominated by large-scale collective motions of bonded and nonbonded groups of atoms. At lower temperatures, simple harmonic vibrations predominate. The transition has been described as a 'glass transition' to emphasize certain similarities between the change in dynamic behavior of individual protein molecules and the changes in viscosity and other properties of liquids when they form a glass. The glass transition may reflect the intrinsic temperature dependence of the motions of atoms in the protein itself, in the bound solvent on the surface of the protein, or it may reflect contributions from both. Protein function is significantly altered below this transition temperature; a fact that can be exploited to trap normally unstable intermediates in enzyme-catalyzed reactions and stabilize them for periods long enough to permit their characterization by high-resolution protein crystallography.     

Translational hydration water dynamics drives the protein glass transition

Experimental and computer simulation studies have revealed the presence of a glass-like transition in the internal dynamics of hydrated proteins at approximately 200 K involving an increase of the amplitude of anharmonic dynamics. This increase in flexibility has been correlated with the onset of protein activity. Here, we determine the driving force behind the protein transition by performing molecular dynamics simulations of myoglobin surrounded by a shell of water. A dual heat bath method is used with which, in any given simulation, the protein and solvent are held at different temperatures, and sets of simulations are performed varying the temperature of the components. The results show that the protein transition is driven by a dynamical transition in the hydration water that induces increased fluctuations primarily in side chains in the external regions of the protein. The water transition involves activation of translational diffusion and occurs even in simulations where the protein atoms are held fixed.     

Simultaneous Measurements of Solvent Dynamics and Functional Kinetics in a Light-Activated Enzyme

Solvent fluctuations play a key role in controlling protein motions and biological function. Here, we have studied how individual steps of the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (POR) couple with solvent dynamics. To simultaneously monitor the catalytic cycle of the enzyme and the dynamical behavior of the solvent, we designed temperature-dependent UV-visible microspectrophotometry experiments, using flash-cooled nanodroplets of POR to which an exogenous soluble fluorophore was added. The formation and decay of the first two intermediates in the POR-catalyzed reaction were measured, together with the solvent glass transition and the buildup of crystalline ice at cryogenic temperatures. We find that formation of the first intermediate occurs below the glass transition temperature (T_g), and is not affected by changes in solvent dynamics induced by modifying the glycerol content. In contrast, formation of the second intermediate occurs above T_g and is influenced by changes in glycerol concentration in a manner remarkably similar to the buildup of crystalline ice. These results suggest that internal, nonslaved protein motions drive the first step of the POR-catalyzed reaction whereas solvent-slaved motions control the second step. We propose that the concept of solvent slaving applies to complex enzymes such as POR.     

Activity,stability and conformational flexibility of seed coat soybean peroxidase

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase superfamily that includes the classical peroxidase, namely horseradish peroxidase (HRP). We have measured the catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(M)) of SBP and that of HRP-C for the oxidation of ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonate)] by hydrogen peroxide at 25 degrees C. We observed that the k(cat) and k(cat)/K(M) values for SBP are much higher than those for HRP-C at all pH values, rendering SBP a more potent peroxidase. This is attributed to the relatively more solvent exposed delta-meso heme edge in SBP. We observed that the maximum catalytic activity and conformational stability of SBP is at pH approximately 5.5. A pH maximum of 5.0 for the catalytic activity of SBP has recently been reported. Estimation of secondary structural elements at various pH values indicated that there is a maximal reduction of beta-strands and beta-turns at pH 5.5 causing the heme to be further exposed to the solvent and increasing the overall conformational flexibility of the protein.     

Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

HIV-1整合酶核心区野生型和F185K突变型活性和溶解性的比较及分子动力学模拟分析

表达纯化了野生型(WT)及F185K突变型HIV-1整合酶核心区蛋白(IN_C),并对二者的溶解性和活性进行了比较．实验结果表明：F185K 突变后IN_C溶解性显著提高,活性有一定程度降低．对WT和F185K IN_C体系进行了1800 ps的分子动力学模拟．模拟结果表明：F185K IN_C功能loop区柔性和蛋白质整体运动性降低,使蛋白质活性降低,F185K突变后盐桥网络的变化驱动了IN_C局部构象改变,引起IN_C表面的部分疏水残基被包埋,亲水残基暴露,相对亲水溶剂可接近面积增大,同时,突变后IN_C与水之间形成氢键的数量增加,与水之间作用加强,以上变化使IN_C溶解性提高．分子动力学模拟与实验结果相吻合．为理解蛋白质溶解性和对蛋白质进行可溶性改造提供了一定的理论依据．     

Hydrogen exchange and the dynamic structure of proteins

Summary In native proteins, buried, labile protons undergo isotope exchange with solvent hydrogens, but the kinetics of exchange are markedly slower than in unfolded polypeptides. This indicates that, whereas buried protein atoms are shielded from solvent, the protein fluctuates around the time average structure and occasionally exposes buried sites to solvent. Generally, hydrogen exchange studies are designed to characterize the nature of the fluctuations between conformational substates, to monitor the shift in conformational equilibria among protein substates due to ligand binding or other factors, or to monitor the major cooperative denaturation transition. In this article, we review the recent reports of hydrogen exchange in proteins, focusing on recent advances in methodology, especially with regard to the implications of the results for the mechanism of hydrogen exchange in folded proteins.     

Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering

The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of biological function at temperatures higher than approximately 25 degrees C (the hyperthermostable enzyme is essentially inactive at room temperature) was associated with a dynamical transition in the anharmonic motions domain. This transition from the nonactive to the enzymatically active conformation involved structurally similar conformational substates in the energy landscape. From the mean-square displacement of the protein atoms, the molecular flexibility and the effective force constants were calculated at different temperature zones. The results showed that the activity increases at higher temperatures where the intramolecular bonds are weakened and the overall rigidity of the protein is decreased. Further temperature increase resulted in significantly increased atomic fluctuations featuring heat denaturation of the protein.     

Identification of dynamical hinge points of the L1 ligase molecular switch

The L1 ligase is an in vitro selected ribozyme that uses a noncanonically base-paired ligation site to catalyze regioselectively and regiospecifically the 5′ to 3′ phosphodiester bond ligation, a reaction relevant to origin of life hypotheses that invoke an RNA world scenario. The L1 ligase crystal structure revealed two different conformational states that were proposed to represent the active and inactive forms. It remains an open question as to what degree these two conformers persist as stable conformational intermediates in solution, and along what pathway are they able to interconvert. To explore these questions, we have performed a series of molecular dynamics simulations in explicit solvent of the inactive–active conformational switch in L1 ligase. Four simulations were performed departing from both conformers in both the reactant and product states, in addition to a simulation where local unfolding in the active state was induced. From these simulations, along with crystallographic data, a set of four virtual torsion angles that span two evolutionarily conserved and restricted regions were identified as dynamical hinge points in the conformational switch transition. The ligation site visits three distinct states characterized by hydrogen bond patterns that are correlated with the formation of specific contacts that may promote catalysis. The insights gained from these simulations contribute to a more detailed understanding of the coupled catalytic/conformational switch mechanism of L1 ligase that may facilitate the design and engineering of new catalytic riboswitches.     

Structural properties of plant and mammalian lipoxygenases. Temperature-dependent conformational alterations and membrane binding ability

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases with major health and political relevance (atherosclerosis, osteoporosis). The crystal structures of various lipoxygenase-isoforms have been reported, and X-ray coordinates for enzyme-ligand complexes are also available. Although the 3D-structures of plant and animal lipoxygenase-isoforms are very similar, recent small-angle X-ray scattering data suggested a higher degree of motional flexibility of mammalian isozymes in aqueous solutions. To explore the molecular basis for these differences we performed dynamic fluorescence measurements that allowed us to study temperature-induced conformational changes arising from three-dimensional fluctuations of the protein matrix. For this purpose, we first investigated the impact of elevated temperature on activity, secondary structure, tertiary structure dynamics and conformational alterations. Applying fluorescence resonance energy transfer we also tested the membrane binding properties of the two lipoxygenase-isoforms, and compared their binding parameters. Taken together, our results indicate that the rabbit 12/15-lipoxygenase is more susceptible to temperature-induced structural alterations than the soybean enzyme. Moreover, the rabbit enzyme exhibits a higher degree of conformational flexibility of the entire protein molecule (global flexibility) and offers the possibility of augmented substrate movement at the catalytic center (local flexibility).     

Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase

Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F1-ATPase.     

Interaction between external medium and haem pocket in myoglobin probed by low-temperature optical spectroscopy

The visible absorption spectra of carbonmonoxymyoglobin in the temperature range 300 to 20 K are reported and compared with the analogous spectra of carbonomonoxyhaemoglobin. The temperature dependence of the zeroth, first and second moment of the observed bands is analysed to obtain information on the local dynamics in the proximity of the haem. Contrary to haemoglobin, the first moment of the observed bands in myoglobin is markedly affected by the solvent composition and its value saturates at temperatures at which the solvent undergoes the glass transition. These data indicate that solvent properties influence the haem pocket stereodynamics in myoglobin; moreover, the different behaviour between myoglobin and haemoglobin suggests that the process should involve the surfaces that are buried in the haemoglobin tetramer and exposed to the solvent in myoglobin, and/or the different protein compressibility.     

Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive "T-state" and a high-affinity active "R-state" with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ("1-Mut") and five ("5-Mut") mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the K(m)(Pyr) is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, k(cat) and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, K(m)(Pyr) approaches the values it has with FBP and becomes almost temperature independent, k(cat) increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.