Structural dynamics, stability and folding of proteins

The present concepts of protein folding in vitro are reviewed. According to these concepts, amino acid sequence of protein, which has appeared a result of evolutionary selection, determines the native structure of protein, the pathway of protein folding, and the existence of free energy barrier between native and denatured states of protein. The latter means that protein macromolecule can exist in either native or denatured state. And all macromolecules in the native state are identical but for structural fluctuations due to Brownian motion of their atoms. Identity of all molecules in native state is of primary importance for their correct functioning. The dependence of protein stability, which is measured as the difference between free energy of protein in native and denatured states, on temperature and denaturant concentration is discussed. The modern approaches characterizing transition state and nucleation are regarded. The role of intermediate and misfolded states in amorphous aggregate and amyloid fibril formation is discussed.     

Energetic basis of structural stability in the molten globule state: alpha-lactalbumin

The denatured states of alpha-lactalbumin, which have features of a molten globule state, have been studied to elucidate the energetics of the molten globule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity increment of alpha-lactalbumin denaturation highly correlates with the degree of disorder of the residual structure of the state. As a result, the denaturational transition of alpha-lactalbumin from the native to a highly ordered compact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entropy of the denaturation of alpha-lactalbumin to compact denatured state are always greater than the enthalpy and entropy of its unfolding. This difference represents the unfolding of the molten globule state. Calorimetric measurements of the heat effect associated with the unfolding of the molten globule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic specificity of the molten globule state, which, in contrast to a protein with unique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and enthalpy of internal interactions. It is concluded that at physiological temperatures the entropy of dehydration is the dominant factor providing stability for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor.     

The three states of globular proteins: acid denaturation.

We describe statistical mechanical theory that aims to predict protein stabilities as a function of temperature, pH, and salt concentration, from the physical properties of the constituent amino acids: (1) the number of nonpolar groups, (2) the chain length, (3) the temperature-dependent free energy of transfer, (4) the pKa's (including those in the native state) and their temperature dependencies. We calculate here the phase diagrams for apomyoglobin and hypothetical variant proteins. The theory captures essential features of protein stability including myoglobin's Tm vs pH as measured by P. L. Privalov [(1979) Advances in Protein Chemistry, Vol. 33, pp. 167-241] and its ionic strength vs pH phase diagram as measured by Y. Goto and A. L. Fink [(1990) Journal of Molecular Biology, Vol. 214, pp. 803-805]. The main predictions here are the following: (1) There are three stable states, corresponding to native (N), compact denatured (C), and highly unfolded (U), with transitions between them. (2) In agreement with experiments, the compact denatured state is predicted to have enthalpy closer to U than N because even though there is considerable hydrophobic "clustering" in C, this nevertheless represents a major loss of hydrophobic contacts relative to configurations (N) that have a hydrophobic "core." (3) C becomes more prominent in the phase diagram with increasing nonpolar content or decreasing chain length, perhaps thus accounting for (a) why lysozyme and alpha-lactalbumin differ in their denatured states, and (b) why shortened Staph nuclease molecules are compact. (4) Of major importance for protein calorimetry is Privalov's observation that the enthalpy of folding, delta H (T, pH) is independent of pH. The theory accounts for this through the prediction that the main electrostatic contribution to stability is not enthalpic; the main contribution is the entropy, mainly due to the different distributions of protons and small ions in the native and denatured states.     

Thermostability of bacteriophage T4 fibritin and its deletion mutants]

The thermal stability of a series of recently obtained mutants of fibritin from bacteriophage T4 (a superhelical fibrous homotrimer with parallel-packed subunits each containing 486 amino acid residues) progressively truncated from the subunit N-end was studied during incubation at 40-90 degrees C in the presence of a surfactant (2% SDS). The mutant fibritins, G, B, C, and E, contained 443, 276, 231, and 120 amino acid residues, respectively. One more truncated mutant (fibritin S1, 108 amino acid residues) was obtained. The 2% SDS-PAGE showed that the migration mobilities of all these proteins corresponded to apparent molecular masses substantially greater than those of the preliminarily heated samples (3 min at 100 degrees C). The heating of the intact fibritin and the mutant G at 50-70 degrees C for 10 min resulted in the formation of a form with an apparent molecular mass higher than 200 kDa. This form probably represented a trimeric protein with a partly denatured N-terminal part. Fibritins B and C were more stable and were only partly denatured into monomers even at 70-90 degrees C. The short mutants E and S1 dissociated into monomers at temperatures from 45 to 50 degrees C. The denaturation of mutants B, C, E, and S1 proceeded in one stage without formation of any intermediate form. The stability of the trimeric molecules of native fibritin under PAGE denaturing conditions and the behavior of the intact protein during heating in the temperature range of 50-70 degrees C might be used for the identification of fibritin intermediate forms upon folding in vivo. The refolding capability was found for fibritin and its mutants denatured by heating at low temperatures in the presence of 2% SDS.     

Thermodynamic investigations of proteins. III. Thermodynamic description of lysozyme.

Standard functions of enthalpy, entropy and the Gibbs energy of native and denatured lysozyme in the range of 0-100 degrees C and pH 1.5-7.0 are represented in three-dimensional projections. The denaturational Gibbs energy change reaches 16 kcal mol-1 at conditions of maximal protein stability (0 degrees C, pH 4.5-7.0) and equals 14.5 kcal mol-1 at 25 degrees C and neutral pH. This result was found to be in agreement with the data reported from guanidine hydrochloride denaturation studies. Partial thermodynamic functions of the conformational and ionizational changes of the protein are obtained from entropy and Gibbs-energy changes in denaturation. The conformational partial entropy and Gibbs-energy change are found to be independent of pH. The pH-dependent partial ionizational entropy and Gibbs-energy changes are induced by normalization of the ionization behaviour of buried groups and cause a decrease of protein stability.     

Thermodynamic investigations of proteins. I. Standard functions for proteins with lysozyme as an example.

A direct method is proposed for obtaining thermodynamic standard functions for native and denatured proteins using experimental data from scanning calorimetry, isothermal calorimetry and potentiometric titrations. The possibility of this approach is demonstrated on the example of lysozyme in the range of pH 1.5-7.0 and temperature 0-100 degrees C. Tests for the validity of the obtained functions of enthalpy and entropy are presented in the form of cyclic processes using experimental data obtained from thermodynamically different pathways. The Gibbs function is checked by comparison with results of an independent method. The methodic problems in determining and checking standard functions for proteins are discussed in detail.     

Theory of cooperative transitions in protein molecules. I. Why denaturation of globular protein is a first-order phase transition

A theory of equilibrium denaturation of proteins is suggested. According to this theory, a cornerstone of protein denaturation is disruption of tight packing of side chains in protein core. Investigation of this disruption is the object of this paper. It is shown that this disruption is an "all-or-none" transition (independent of how compact is the denatured state of a protein and independent of the protein-solvent interactions) because expansion of a globule must exceed some threshold to release rotational isomerization of side chains. Smaller expansion cannot produce entropy compensation of nonbonded energy loss; this is the origin of a free-energy barrier (transition state) between the native and denatured states. The density of the transition state is so high that the solvent cannot penetrate into protein in this state. The results obtained in this paper make it possible to present in the following paper a general phase diagram of protein molecule in solution.     

Correlation between changes in nuclear magnetic resonance order parameters and conformational entropy: molecular dynamics simulations of native and denatured staphylococcal nuclease

Recent work has suggested that changes in NMR order parameters may quantitatively reflect changes in the conformational entropy of a protein ensemble. The extent of the mathematical relationship between local entropy changes as seen by NMR order parameters and the full protein entropy change is a complex issue. As a step towards a fuller understanding of this problem, molecular dynamics calculations of both native and denatured staphylococcal nuclease were performed. The N-H bond vector motion, in both explicit and implicit solvent, was analyzed to estimate local and global entropy changes. The calculated N-H bond vector order parameters from simulation agreed on average with experimental values for both native and denatured structures. However, the inverted-U profile of order parameters versus residue number observed experimentally for denatured nuclease was only partially reproduced by simulation of compact denatured structures. Comparisons made across the full set of simulations revealed a correlation between the N-H order parameter-based conformational entropy change and the total quasiharmonic-based conformational entropy change between the native and denatured structures. The calculations showed that about 25% of the total entropy change was reflected by changes in simulated S2 values. This result suggests that NMR-derived order parameters may be used to provide a reasonable estimate of the total conformational entropy change on protein folding.     

Fluorescence ratio intrinsic basis states analysis: a novel approach to monitor and analyze protein unfolding by fluorescence

Fluorescence ratio intrinsic basis states analysis (FRIBSTA) is a novel method allowing quantitative estimation of the stability of proteins in aqueous solution as a function of temperature. In FRIBSTA emission fluorescence spectra are repeatedly recorded while ramping temperature from < or =-15 to > or =100 degrees C. Subsets of these are identified as reference spectra of the protein in either its folded or in its heat denatured configuration. Each reference spectrum of both sets is normalized by its own integrated fluorescence intensity to give a fractional area spectrum. Linear extrapolations of these normalized reference spectral shapes over the entire temperature range of measurement are then used to deconvolute each experimental emission spectrum to give a fraction of emission from native state and a fraction from denatured state. Additionally, the integrated emission fluorescence intensity for the native configuration is fitted and extrapolated over the temperature range of measurement. Division of the deconvoluted native integrated fluorescence intensity by the fitted-extrapolated integrated emission fluorescence intensity yields the fraction folded. The free energy functions derived from fraction unfolded are presented for beta-lactoglobulin and phosphoglycerate kinase. According to these results both proteins are considerably less stable than heretofore assumed at ambient temperatures and partially denatured at temperatures < or =0 degrees C. The method is employed to study the effect of denaturants on these proteins as well. The major usefulness of FRIBSTA is that one can directly measure the protein stability at ambient and subambient temperatures in the absence of denaturants rather than predicting it by extrapolation from heat denaturation data.     

Coarse-grained strategy for modeling protein stability in concentrated solutions. II: phase behavior

We use highly efficient transition-matrix Monte Carlo simulations to determine equilibrium unfolding curves and fluid phase boundaries for solutions of coarse-grained globular proteins. The model we analyze derives the intrinsic stability of the native state and protein-protein interactions from basic information about protein sequence using heteropolymer collapse theory. It predicts that solutions of low hydrophobicity proteins generally exhibit a single liquid phase near their midpoint temperatures for unfolding, while solutions of proteins with high sequence hydrophobicity display the type of temperature-inverted, liquid-liquid transition associated with aggregation processes of proteins and other amphiphilic molecules. The phase transition occurring in solutions of the most hydrophobic protein we study extends below the unfolding curve, creating an immiscibility gap between a dilute, mostly native phase and a concentrated, mostly denatured phase. The results are qualitatively consistent with the solution behavior of hemoglobin (HbA) and its sickle variant (HbS), and they suggest that a liquid-liquid transition resulting in significant protein denaturation should generally be expected on the phase diagram of high-hydrophobicity protein solutions. The concentration fluctuations associated with this transition could be a driving force for the nonnative aggregation that can occur below the midpoint temperature.     

Study of the interaction of DNA and acridine orange by various optical methods

The degree of binding of acridine orange to DNA, native or denatured, has been determined by equilibrium dialysis in 0.1M and 0.001M NaCl at 20°. The nature of the binding process has been investigated by studying various optical properties of the dye–DNA complexes and by relating them to the binding ratio. All these properties were found to vary quantitatively and qualitatively according to the successive stages of the process. These stages were assumed to be a strong binding of intercalated monomers followed by formation of bound dimers and finally by external binding of aggregates of native DNA. Absorption spectra of the complexes could be interpreted on that basis. Circular dichroism spectra were resolved into components: one band for intercalated monomers without interactions, two excition splittings for interacting monomers and bound dimers, respectively, weak bands and exciton splitting for external aggregates. The fluorescence intensity was greatly enhanced in intercallated monomers; its quenching at higher binding ratio was quantitatively related to dimer fixation. The value of the anisotropy of fluorescence at low binding ratio suggested a limited mobility of intercalated monomers; the decrease of polarization at higher binding was attributed to energy transfer between monomers. Electric dichroism displayed by the complexes in the dye absorption bands indicated an orientation of the bound molecules quite parallel to the base rings at low binding. In the range of fixation of dimers and external molecules, the dichroism was lower but still indicated an important degree of ordering.     

Thermal denaturation and gelation of rubisco: effects of pH and ions

In order to understand the mechanism of thermal gelation of rubisco, its native and heat denatured states were characterized by absorbance, fluorescence and circular dichroïsm spectroscopies as well as by differential scanning calorimetry in the presence of various salts. It appears that during the denaturation process, divalent anions are released while divalent cations are fixed by the protein, while it is disorganized and while the environment of its aromatic chromophores becomes more hydrophilic. The pH transition of gelation is shifted 1–2 pH units higher than the transition of denaturation temperature which occurs near the isoelectric point of the native molecule. This shift probably corresponds to the breaking of saline bridges within the protein molecule. Finally, a large effect of divalent cations on the phase diagram indicates that a particular denatured state is attained when these cations are in the denaturation medium.     

Urea‐temperature phase diagrams capture the thermodynamics of denatured state expansion that accompany protein unfolding

We have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea‐induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea–temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two‐state character. Global analysis of the urea–temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of and that define a complex temperature dependence of the m‐value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea‐denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ～40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions.     

Temperature-, electric field- and solute-induced percolation in water-in-oil microemulsions

We report investigations on the percolation of the aqueous phase in water-in-oil microemulsions, comparing systems stabilized by ionic AOT and non-ionic Igepal amphiphiles. First, we briefly review the opposite effect of temperature on the two systems and compare electric conductivity with viscosity data. In the second part, we show that percolation can be induced by high electric fields resulting in a shift of the percolation curve. The electric field measurements allow to investigate the dynamics of clustering of the water droplets to form a network of percolating channels. We examine the slow build-up and the fast decay of the percolating structure, monitoring simultaneously electric conductivity and electric birefringence. In the third part we discuss the effect of some solutes on the percolation curve, especially of small molecules which act as protein denaturants and of native and denatured proteins like methemoglobin, chymotrypsin and gelatin. The spectroscopic determination of the dimerization of hemin, released from denatured hemoglobin, reflects the incorporation of the hemin monomers in the surfactant monolayer. In the gelatin system time resolved electric birefringence shows that even at low concentrations it is the macromolecule which determines the structure of the aqueous domain. In the appendix, a simple estimate of the intrinsic Kerr-constant is given for microemulsion droplets deformed in an electric field.     

Thermal denaturation of cytochromes c of horse cow, and Candida krusei in aqueous guanidine hydrochloride.

Thermal denaturation of cytochromes c of horse, cow, and Candida krusei in aqueous guanidine hydrochloride in the neutral pH region was studied by means of absorption and optical rotation measurements. The values of standard free energy change upon denaturation were estimated over the temperature range from 3 to 51 degrees C. Large differences in the heat capacity of the native and denatured states amounting to several kcal/mol-deg were obtained for all three kinds of cytochromes c. These lead to a change in the sign of both the enthalpy and entropy change of denaturation, with maximum stability of the native state at 12 degrees C for horse and bovine cytochromes c and at 9 degrees C for Candida krusei.     

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.     

The kinetics and thermodynamics of reversible denaturation of crystalline soybean trypsin inhibitor

Crystalline soybean trypsin inhibitor protein undergoes denaturation on heating which is reversed on cooling. In the range of temperature of 35 to 50 degrees C. a solution of the protein consists of a mixture of native and denatured forms in equilibrium with each other. The equilibrium is only slowly established and its final value at any temperature is the same whether a heated, denatured solution of the protein is cooled to the given temperature or whether a fresh solution is raised to that temperature. The kinetics of reversible denaturation of the soybean protein as well as the reversal of denaturation is that of a reversible unimolecular reaction, each process consisting at a given temperature of the same two simultaneous reactions acting in opposite directions. The experimental data on the effect of temperature on the velocity and the equilibrium constants of the opposing reaction were utilized in evaluating the reaction energies and activation energies. The reaction energies for denaturation were found to be as follows:- Change in total heat of reaction DeltaH = 57,000 calories per mole Change in entropy of reaction DeltaS = 180 calories per degree per mole The heat of activation DeltaH(1) (double dagger) for denaturation = 55,000 The heat of activation DeltaH(2) (double dagger) for the reversal of denaturation = -1900 The entropy DeltaS(1) (double dagger) for denaturation = 95 The entropy DeltaS(2) (double dagger) for reversal of denaturation = -84