蛋白质变性机理与变性时的热力学参数研究进展

生物大分子是近年来生命科学的研究热点和难点之一,而对蛋白质变性的研究有助于深刻揭示生命现象的机理.利用光谱学和热力学可以分别从微观和宏观角度对蛋白质变性进行研究,并由此得到表征蛋白质变性的热力学参数.这对深入了解蛋白质的折叠与伸展、变性机理、结构稳定性及生命体的新陈代谢等问题具有很大意义.近年来,国内外学者在此方面做了大量的工作,主要涉及蛋白质在水溶液中的变性机理、在有变性剂存在下水溶液中的变性机理及在含有其它物质水溶液中的变性机理.用来表征蛋白质变性的热力学参数有热容、变性自由能、变性焓和变性熵等.本文对这些研究进行了概述.     

Microcalorimetric study of thermal unfolding of lysozyme in water/glycerol mixtures: an analysis by solvent exchange model

Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by differential scanning microcalorimetry and related to difference in preferential solvation of native and denatured states. In particular, the thermal denaturation of a model system formed by lysozyme dissolved in water in the presence of the stabilizing cosolvent glycerol has been considered. Transition temperatures and enthalpies, heat capacity, and standard free energy changes have been determined when applying a two-state denaturation model to microcalorimetric data. Thermodynamic parameters show an unexpected, not linear, trend as a function of solvent composition; in particular, the lysozyme thermodynamic stability shows a maximum centered at water molar fraction of about 0.6. Using a thermodynamic hydration model based on the exchange equilibrium between glycerol and water molecules from the protein solvation layer to the bulk, the contribution of protein-solvent interactions to the unfolding free energy and the changes of this contribution with solvent composition have been derived. The preferential solvation data indicate that lysozyme unfolding involves an increase in the solvation surface, with a small reduction of the protein-preferential hydration. Moreover, the derived changes in the excess solvation numbers at denaturation show that only few solvent molecules are responsible for the variation of lysozyme stability in relation to the solvent composition.     

Protein folding kinetics: barrier effects in chemical and thermal denaturation experiments

Recent experimental work on fast protein folding brings about an intriguing paradox. Microsecond-folding proteins are supposed to fold near or at the folding speed limit (downhill folding), but yet their folding behavior seems to comply with classical two-state analyses, which imply the crossing of high free energy barriers. However, close inspection of chemical and thermal denaturation kinetic experiments in fast-folding proteins reveals systematic deviations from two-state behavior. Using a simple one-dimensional free energy surface approach we find that such deviations are indeed diagnostic of marginal folding barriers. Furthermore, the quantitative analysis of available fast-kinetic data indicates that many microsecond-folding proteins fold downhill in native conditions. All of these proteins are then promising candidates for an atom-by-atom analysis of protein folding using nuclear magnetic resonance.1 We also find that the diffusion coefficient for protein folding is strongly temperature dependent, corresponding to an activation energy of approximately 1 kJ.mol-1 per protein residue. As a consequence, the folding speed limit at room temperature is about an order of magnitude slower than the approximately 1 micros estimates from high-temperature T-jump experiments. Our analysis is quantitatively consistent with the available thermodynamic and kinetic data on slow two-state folding proteins and provides a straightforward explanation for the apparent fast-folding paradox.     

Spectral researches on interaction of laser radiation with targets from titanium oxides

Spectral characteristics and kinetic processes of the laser-induced plasma used for the growth of films from titanium oxides were investigated. The experiments were carried out in vacuum, oxygen, and an oxygen radiofrequency discharge plasma and its afterglow. A collision-radiative model describing the state-to-state kinetics of titanium atoms and ions at various stages of flight of laser-induced plasma was proposed. It was shown that the conditions of local thermodynamic equilibrium are disturbed because of a fast gas-dynamic expansion of plasma. Nonetheless, the distribution functions of titanium atoms and ions over excited states are described by the Boltzmann formulas with different temperatures.     

Binary homogeneous nucleation in water-succinic acid and water-glutaric acid systems

Binary homogeneous nucleation of water-succinic acid and water-glutaric acid systems have been investigated. The numerical approach was based on the classical nucleation theory. Usually, nucleation is discussed in terms of kinetics, but the thermodynamics involved is undoubtedly equally important. In this paper we studied the above mentioned binary systems giving a quantitative insight into the nucleation process and a detailed consideration of the thermodynamics involved. Both diacids in study are in solid state at room temperature. They behave in environment according to their liquid state properties because of the absence of crystalline lattice energies, and therefore their subcooled liquid state thermodynamics have to be considered. The lack of consistent thermodynamic data for pure organic components and their aqueous solutions represent a high source of uncertainty. However, the present simulations indicate that in atmospheric conditions these binary systems will not form new particles.     

有机配体Schiff碱4-(2-羟基苯基)-亚氨基-戊-2-酮的从头算研究

本文通过从头算HF／6－31G（d）方法,对Schiff4－（2－羟基苯基）－亚氨基－戊－2－酮 进行了理论研究,提供了该化合物两种互变异构体的几何构型参数、电子结构、IR光谱性 、偶极矩数据,并借助热、动力学手段,分析了两种互变异构体的异构平衡过程。计算结果表明：（1）从几何构型、电子 结构和相对能量的角度考虑,由于较强的分子内氢键作用和较大的共轭体系,亚胺烯醇式更为稳定。（2）从分子极性的角度考虑,烯胺酮式具有较大的偶极矩,其较强的分子间作用力有利形成晶体,因而烯胺酮式以晶体的形式存在。（3）由烯胺酮式向亚胺烯醇式转化的互变异构反应是热力学自发反应,但受到较大活化能的控制,是一个动力学稳定体系。分析了极性溶剂存在的互变异构反应是热力学自发反应,但受到较大活化能的控制,是一个动力学稳定体系。分析了极性溶剂存在将有利于反应发生且标题化合物以亚胺烯醇式存在于极性溶剂中的作用。以上结论均与实验研究结果相符。     

Kinetics of proton transfer in a green fluorescent protein: A laser-induced pH jump study

The sub-millisecond protonation dynamics of the chromophore in S65T mutant form of the green fluorescent protein (GFP) was tracked after a rapid pH jump following laser-induced proton release from the caged photolabile compoundo-nitrobenzaldehyde. Following a jump in pH from 8 to 5 (which is achieved within 2 μs), the fluorescence of S65T GFP decreased as a single exponential with a time constant of ∼90 μs. This decay is interpreted as the conversion of the deprotonated fluorescent GFP chromophore to a protonated non-fluorescent species. The protonation kinetics showed dependence on the bulk viscosity of the solvent, and therefore implicates bulk solvent-controlled protein dynamics in the protonation process. The protonation is proposed to be a sequential process involving two steps: (a) proton transfer from solvent to the chromophore, and (b) internal structural rearrangements to stabilize a protonated chromophore. The possible implications of these observations to protein dynamics in general is discussed     

Hydrogen-bonding effects on the formation and lifetimes of charge-separated States in molecular triads

Photoinduced electron transfer in two molecular triads comprised of a triarylamine donor, a d(6) metal diimine photosensitizer, and a 9,10-anthraquinone acceptor was investigated with particular focus on the influence of hydrogen-bonding solvents on the electron transfer kinetics. Photoexcitation of the ruthenium(II) and osmium(II) sensitizers of these triads leads to charge-separated states containing an oxidized triarylamine unit and a reduced anthraquinone moiety. The kinetics for formation of these charge-separated states were explored by using femtosecond transient absorption spectroscopy. Strong hydrogen bond donors such as hexafluoroisopropanol or trifluoroethanol cause a thermodynamic and kinetic stabilization of these charge-separated states that is attributed to hydrogen bonding between alcoholic solvent and reduced anthraquinone. In the ruthenium triad this effect leads to a lengthening of the lifetime of the charge-separated state from ～750 ns in dichloromethane to ～3000 ns in hexafluoroisopropanol while in the osmium triad the respective lifetime increases from ～50 to ～2000 ns between the same two solvents. In both triads the lifetime of the charge-separated state correlates with the hydrogen bond donor strength of the solvent but not with the solvent dielectric constant. These findings are relevant in the greater context of solar energy conversion in which one is interested in storing light energy in charge-separated states that are as long-lived as possible. Furthermore they are relevant for understanding proton-coupled electron transfer (PCET) reactivity of electronically excited states at a fundamental level because changes in hydrogen-bonding strength accompanying changes in redox states may be regarded as an attenuated form of PCET.     

On the estimation of stability parameters from heat-induced conformational transition curves of proteins

A method is suggested to determine valid and authentic values of thermodynamic stability parameters of proteins from their heat-induced conformational transition curves. We show (a) that the estimate of ΔH_m ^van, the enthalpy change on denaturation at T_m, the midpoint of denaturation, is significantly less than ΔH_m ^cal, the value obtained by the calorimetric measurements, if the analysis of the conformational transition curve uses the conventional method which assumes a linear temperature-dependence of the pre- and post-transition baselines; and (b) that there exists an excellent agreement between ΔH_m ^van and ΔH_m ^cal values of proteins, if the analysis of thermal denaturation curves assumes that the temperature-dependence of pre- and post-transition baselines is described by a parabolic function. The latter analysis is supported by our observations that the temperaturedependencies of the absorption and circular dichroism properties of protein groups are indeed nonlinear. It is observed that the estimate of ΔC_p, the constant-pressure heat capacity change is independent of the model used to describe the temperaturedependence of the pre- and post-transition baselines. An important conclusion is that for proteins which exhibit a two-state character, all stability parameters are measured with the same error as that observed with a calorimeter.     

Electrochemistry of vitamin B12: Part IX. The Co(III)/Co(II) couple in non-aqueous solvents

The thermodynamic and kinetic characteristics of the Co(III)/Co(II) couple are investigated as a function of the solvent and thesixth axial ligand. With solvatocobalamins the thermodynamics of the Co(III)/Co(II) oxido-reduction reaction essentially reflect the strength of the Co(III)-solvent interaction. The order of the solvents in this connection appears to beH₂O>DMSO>ROH. The kinetics of the reaction parallels and amplifies the thermodynamic effects: the stronger the Co(III)-solvent interaction, the slower the reaction. While chloride and acetate ions appear as very weak ligands in water, they are much stronger in aliphatic alcohols and DMSO. The variations of the thermodynamic factors observed as a function of the solvents and the ligands are rationalized in terms of interaction of the solvent with Co(II) and with the anionic ligand and of the affinity of the ligand with Co(III). The kinetics of the Co(III)/Co(II) oxido-reduction reaction, involving both electron transfer and bond-breaking, again parallels and amplifies the thermodynamic effect. The stretching of the Co-ligand bond and the solvation of the anionicligands appear as the main kinetic factors.     

Modification of the thermal unfolding pathways of myoglobin upon drug interaction in different aqueous media

In this work, we have analyzed the influence of two structurally related phenothiazine drugs, promazine and triflupromazine hydrochlorides, when bound to myoglobin, a model protein, and how the drug concentration and solution conditions may affect the denaturation process of this protein. In this manner, we derive the thermodynamic quantities of the unfolding process by using a spectroscopic technique such as UV-vis spectroscopy at different drugs concentrations and at pH 2.5, 5.5, and 9.0. To do this, a thermodynamic model was used which included experimental data corresponding to the pre- and post-transition into the observable transition. It has been found that both drugs play a destabilizing role for the protein, at least at low concentrations. In addition, at acidic pH and higher drug concentrations, a stabilizing effect can be observed, which may be related to the formation of some type of protein refolding, subsequent aggregation, or both. The reason for this behavior has been suggested to be the different protein conformations at acidic pH, the increase of solvent-exposed hydrophobic and hydrophilic residues after denaturation and/or binding, and the different strength of drug-protein interactions when changing the solution conditions. For this reason, thermodynamic quantities such as Gibbs energies, DeltaG, and entropies of unfolding, DeltaS(m), increase as the solution pH increases provided that additional solvent-exposed hydrophobic residues are present, which were previously buried at room temperature. Moreover, the larger binding affinity at pH 9.0 due to enhanced electrostatic interactions between protein and drug molecules (drug and protein differ in their net electrical charge) additionally collaborates to this residue exposition to solvent as a consequence of the alteration of protein conformation as due to drug binding. Comparison of thermodynamic data between promazine and triflupromazine hydrochlorides also shows that drug-protein affinity and hydrophobicity also affect the thermodynamic denaturation parameters.     

Reversed-phase retention thermodynamics of pure-water mobile phases at ambient and elevated temperature

The use of pure water at superheated temperatures, between 100 and 200 degrees C, as a mobile phase for reversed-phase separations is explored. The thermodynamics of the retention process at low temperature (15-55 degrees C) are compared to the thermodynamics at elevated temperature (125-175 degrees C). Significant differences in the enthalpy of the retention process are observed between the two temperature ranges. This is possibly due to changes in the hydrogen-bond network of the pure-water mobile phase, which would change the solvation, and therefore retention, of non-polar solutes. The change in thermodynamic values between the two temperature regions invalidates extrapolation of retention as a function of temperature between the two temperature regions for the prediction of room-temperature pure-water retention factors. The thermodynamic changes observed as the temperature is increased are similar to those seen when mobile phase composition is changed (by adding organic modifier) at constant temperature.     

The McMillan–Mayer framework and the theory of electrolyte solutions

In electrolyte thermodynamics one often speaks of two thermodynamic frameworks; the Lewis–Randall framework (characterised by temperature, pressure, and mole numbers) and the McMillan–Mayer framework (characterised by temperature, total volume, solute mole numbers, and solvent chemical potential). However, there is only one framework in thermodynamics; the apparent difference between the two ‘frameworks’ is, in electrolyte thermodynamics, due to the change in the pressure caused by the charging process at constant volume and solvent chemical potential.The so-called McMillan–Mayer framework is set in the context of the classical thermodynamics and the use of it is examplified by the Debye–Hckel theory. The so-called McMillan–Mayer framework is superfluous when the thermodynamics of the electrolyte solutions is described by the Helmholtz energy functions.     

Solvent Effect on The Equilibrium and Rate Constant of the Tautomeric Reaction in Nexium,Skelaxin, Aldara and Efavirenz Drugs: A Dft Study

Some drugs have two tautomeric structures in the liquid state and the tautomeric equilibrium is formed between these structures. The reaction rate and equilibrium constant of this reaction varies in different solvents. Thus, the determination of the appropriate solvent to separate tautomers is important. In this research, we used the DFT level of B3LYP and the 6-311++G** basis set to obtain the proper solvent for Nexium, Skelaxin, Aldara and Efavirenz drugs. All calculations were made above the melting point of the mentioned drugs, because these drugs are solid at room temperature. The solvent effect is included in the calculation utilizing the polarizable continuum model PCM. The transition state of the tautomeric reaction is determined using the quadratic synchronous transit (QST2) method. The geometry, energy, and dipole moment of transition states are analyzed in different solvent. The root mean square deviation (RMSD) analysis is performed to determine the degree of a structure deviation of tautomer 1 and tautomer 2 from the transition state in different solvents. It is found that the RMSD value for tatumer1 is higher than that for tautomer 2 in all studied drugs. The proper solvent for the separation of tautomers is determined from the analysis of thermodynamics and kinetics.     

The temperature-induced denaturation of small globular proteins as a first-order phase transition of ‘crystal molecules’

A general feature of temperature-induced reversible denaturation of small globular proteins is its all-or-none character. This strong cooperativity leads to think that protein molecules, possessing only two accessible thermodynamic states, the native and the denatured one, resemble ‘crystal molecules’ that melt at raising temperature. An analysis, grounded on mean field theory, allows to conclude that the two-state transition is a first-order phase transition. The implication of this conclusion are briefly discussed.     

Entropic barriers in nanoscale adhesion studied by variable temperature chemical force microscopy

Intermolecular interactions drive the vast majority of condensed phase phenomena from molecular recognition to protein folding to particle adhesion. Complex energy barriers encountered in these interactions include contributions from van der Waals forces, hydrogen bonding, and solvent medium. With the spectacular exception of hydrophobic interactions, contributions from the medium are usually considered secondary. We report a variable temperature force microscopy study of the interactions between several hydrogen bonds in different solvents that challenges this point of view. Surprisingly, we observed an increase in the strength of the interaction between carboxylic acid groups in ethanol as the temperature increased. Moreover, when we switched to a nonpolar solvent we observed the opposite behavior: The binding force decreased as the temperature increased. Kinetic model of bond dissociation provided quantitative interpretation of our measurements. We attributed the observed phenomena to a large entropic contribution from the ordered solvent layers that are forming on the probe and sample surfaces upon detachment. The observed reversal in the force vs temperature trend is a manifestation of a transition between thermodynamic and kinetic regimes of unbinding predicted by the model. Our results indicate that entropic barriers dominated by the interactions of solvent molecules with the surface exist in a much wider variety of systems than previously thought.     

Hydrogen exchange mass spectrometry of bacteriorhodopsin reveals light-induced changes in the structural dynamics of a biomolecular machine

Many proteins act as molecular machines that are fuelled by a nonthermal energy source. Examples include transmembrane pumps and stator-rotor complexes. These systems undergo cyclic motions (CMs) that are being driven along a well-defined conformational trajectory. Superimposed on these CMs are thermal fluctuations (TFs) that are coupled to stochastic motions of the solvent. Here we explore whether the TFs of a molecular machine are affected by the occurrence of CMs. Bacteriorhodopsin (BR) is a light-driven proton pump that serves as a model system in this study. The function of BR is based on a photocycle that involves trans/cis isomerization of a retinal chromophore, as well as motions of transmembrane helices. Hydrogen/deuterium exchange (HDX) mass spectrometry was used to monitor the TFs of BR, focusing on the monomeric form of the protein. Comparative HDX studies were conducted under illumination and in the dark. The HDX kinetics of BR are dramatically accelerated in the presence of light. The isotope exchange rates and the number of backbone amides involved in EX2 opening transitions increase roughly 2-fold upon illumination. In contrast, light/dark control experiments on retinal-free protein produced no discernible differences. It can be concluded that the extent of TFs in BR strongly depends on photon-driven CMs. The light-induced differences in HDX behavior are ascribed to protein destabilization. Specifically, the thermodynamic stability of the dark-adapted protein is estimated to be 5.5 kJ mol(-1) under the conditions of our work. This value represents the free energy difference between the folded state F and a significantly unfolded conformer U. Illumination reduces the stability of F by 2.2 kJ mol(-1). Mechanical agitation caused by isomerization of the chromophore is transferred to the surrounding protein scaffold, and subsequently, the energy dissipates into the solvent. Light-induced retinal motions therefore act analogously to an internal heat source that promotes the occurrence of TFs. Overall, our data highlight the potential of HDX methods for probing the structural dynamics of molecular machines under "engine on" and "engine off" conditions.     

Studies on gelation of soybean globulin solutions

Thermal denaturation of soybean globulin fraction (SBGF) in diluted solution (protein concentration 0.15–0.63%) has been studied by the method of differential adiabatic scanning calorimetry. SBGF thermograms have two maxima. The low temperature maximum is consistent with denaturation of 7S component, while the high temperature maximum with denaturation of 11S components of this fraction. In the investigated range of protein concentrations the thermodynamic parameters (temperature and enthalpy) of denaturation of SBGF and its main components are constant. This fact suggests that differential adiabatic scanning calorimetry gives information purporting a change in the protein state at molecular level. The temperatures and enthalpies of denaturation of the main SBGF components linearly rise with increase of NaCl concentration. The slope of dependences of denaturation temperature on salt concentration,K _s, is extremely large (nearly 20 K · l/mole). The elementary thermodynamic theory of lyotropic effects in thermal denaturation of proteins has been developed based on the two-state model and linear approximation of protein-salt interactions by means of the corresponding second virial coefficient. It shows that the dependences of thermodynamic parameters of thermal denaturation on salt concentration should be linear in the initial section. This conclusion is consistent with the experiment. The differences of enthalpies and entropies of transferring denatured and native forms of the main SBGF components from water into NaCl solution have been determined. They are positive and their quantity increases linearly with salt concentration. This fact is consistent with the concept to the effect that the main factor of salt influence on thermal denaturation of SBGF is confined to a decrease of protein hydration. The effect of protein nature on the quantity of lyotropic effect in thermal denaturation has been considered. Using simple considerations as a basis, the dependence of the ratio betweenK _s and the denaturation temperature in water has been obtained, which characterizes the lyotropic effect, on the molar fraction of hydrophobic residues in the protein molecule. This dependence is linear and the lyotropic effect rises with increase in the content of hydrophobic residues. It is satisfactorily consistent with the experimental data on NaCl effect on thermal denaturation temperature for ichthyocol gelatin, ribonuclease, lysozyme, 7S and 11S SBGF components. An extraordinary strong influence of NaCl on thermal denaturation temperatures for the main SBGF components can be accounted for by a relatively high content of hydrophobic residues.