Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry: evidence for noncooperative nucleotide binding.

We characterized the thermodynamics of binding reactions of nucleotides ADP and ATPgammaS (a nonhydrolyzable analog of ATP) to GroEL in a temperature range of 5 degrees C to 35 degrees C by isothermal titration calorimetry. Analysis with a noncooperative binding model has shown that the bindings of nucleotides are driven enthalpically with binding constants of 7x103 M-1 and 4x104 M-1 for ADP and ATPgammaS, respectively, at 26 degrees C and that the heat capacity change DeltaCp is about 100 cal/mol.K for both the nucleotides. The stoichiometries of binding were about 8 and 9 molecules for ADP and ATPgammaS, respectively, per GroEL tetradecamer at 5 degrees C, and both increased with temperature to reach about 14 (ADP) and 12 (ATPgammaS) for both nucleotides at 35 degrees C. The absence of initial increase of binding heat as well as Hill coefficient less than 1.2, which were obtained from the fitting to the model curve by assuming positive cooperativity, showed that there was virtually no positive cooperativity in the nucleotide bindings. Incorporating a difference in affinity for the nucleotide (ADP and ATPgammaS) between the two rings of GroEL into the noncooperative binding model improved the goodness of fitting and the difference in the affinity increased with decreasing temperature.

Intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in goat alpha-lactalbumin.

Goat alpha-lactalbumin shows two septral changes at acid pH. One of these corresponds to the acid denaturation, while the other has been regarded as a result of the intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in the native molecule. In order to distinguish between these spectral changes and to investigate the perturbation caused by the ionizable groups, the absorption changes with a change in pH have been followed by the stopped-flow pH-jump method. Only the acid denaturation can be observed kinetically while the perturbation is too rapid to follow by the kinetic method. Extrapolations of the time-dependent absorption changes to zero time in the unfolding and refolding pH jumps give the apparent titration curves of the ionizable groups that participate in the perturbations in the native and in the acid-denatured molecule. The perturbation of the native protein is induced by the protonation of at least two ionizable groups, while the acid-denatured molecule does not show the perturbation because of the loss of the unique spatial arrangement of the ionizable groups around tryptophans. The results of circular dichroism measurements and the comparison with known results of the charge perturbation of lysozyme are also discussed.

Kinetic correlations between the disulfide bond reduction and the induced conformational change of proteins.

Kinetic correlations between the disulfide bond reduction in excess dithioerythritol and the induced conformational change were studied on two proteins, bovine alpha-lactalbumin and soybean trypsin inhibitor, at 25 degrees C and pH 8.0-8.5 by measuring the absorbance of oxidized dithioerythritol at 310 nm and the ellipticity at 270 nm, respectively. With alpha-lactalbumin, in the absence of guanidine hydrochloride (Gdn x HCl) or in dilute Gdn x HCl, the kinetics for the bond reduction and the conformational change were both of a biphasic type. The fast phase was complete within a few seconds and was associated with the reduction of some of the disulfide bonds and with almost complete loss of the tertiary structure. The slow phase was associated with the reduction of other disulfide bonds. In concentrated Gdn x HCl, the kinetics of both processes were observed as a single phase, the rate of which was similar to that of the slow phase in the absence of Gdn x HCl or in dilute Gdn x HCl. In all cases studied, the rate of the bond reduction was similar to that of the conformational change induced. By correcting the change in absorbance at 310 nm to a contribution from the protein due to the conformational change, the number of bonds which are reduced in the fast phase in the absence of Gdn x HCl was determined to be 1.0-1.1. It was shown, taking observations of others and theoretical results into account, that the bond reduced in the fast phase might be the one between Cys 6 and Cys 120. On the other hand, on of two bonds of soybean trypsin inhibitor in the native form was reduced in the fast phase without any loss of the tertiary structure, and the other was reduced in the slow phase. Considering the results of other researchers, it was concluded that the bond reduced in the fast phase of the inhibitor is a 136-145 bond.

Thermodynamic characterization of partially denatured states in the denaturation process of bovine alpha-lactalbumin by inorganic denaturants.

In an attempt to understand the specific effect of inorganic protein denaturants, lithium cation and perchlorate anion, upon the molecular conformation of bovine alpha-lactalbumin and to characterize the denatured states of the protein and the denaturation processes, themodynamic studies on the reversible unfolding of the protein in the presence of lithium chloride, lithium perchlorate and sodium perchlorate were made by means of circular dichroism and ultraviolet absorption measurements. The denaturation reaction caused by lithium chloride was found to take place in a three-state type, while that caused by the two perchlorates in a two-state type. The latter produces the same denatured state as the acid does on the protein, the state where the helical structures remain unchanged. The former produces two kinds of the denatured state, one being a less unfolded state than the acid denatured one and the other a fully unfolded state which is identical with the finally denatured state induced by organic denaturants such as guanidinium chloride, guanidinium thiocyanate and urea.

Equilibrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride (IV): dependence of the N equilibrium A transconformation on the temperature.

The reversible unfolding from the native (N) state to the acid (A) state of alpha-lactalbumin by guanidine-HCl (0.8-2.0 M) was studied at 10-35 degrees C by means of difference spectra and pH-jump measurements. At each temperature, all points plotted as the logarithmic equilibrium constant log KNA of the N equilibrium A process against pH could fall on a curve independent of the denaturant concentration by shifting each point along the log KNA axis, where the shift factor f did not depend on temperature. Moreover, by shifting the points at each temperature along the log (KNA/f) axis, a master curve, independent of both temperature and the denaturant concentration, could be obtained for the pH-dependence of log KNA. From the dependence of the logarithmic rate constants on pH, master curves independent of both temperature and the denaturant concentration could also be made for the N leads to A and the A leads to A processes, where A mean the activated state. The results show the two-state character of the N equilibrium A process. The enthalpy changes and the differences in heat capacity for the N equilibrium A, N equilibrium A and A equilibrium A processes were determined from the accurate measurements of temperature dependence of the unfolding at pH 4.3 and 1.0 M guanidine-HCl. The results show that the disruption of hydrophobic interaction is caused mainly in the A leads to A process, while most of the changes in the pK values of the ionizing groups are caused in the N leads to A process.

Equilbrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride (II).

The reversible unfolding of alpha-lactalbumin by guanidine hydrochloride, was studied at 25.0 degrees C in a relatively low concentration range of the denaturant (0.80-2.00 mol/l) by means of difference spectra and pH-jump measurements. The unfolding was shown to occur between two states, N and D, because apparent rate-constants of the unfolding and the refolding reactions depended only on pH. All curves plotted as the logarithmical equilibrium constant log KD against pH could fall on the same base curve by shifting each curve along the log KD axis. From the dependence of the logarithmic rate constant on pH, master curves could also be made for the forward and the backward reactions. The dependence of these master curves on pH indicates that the groups affecting the pH dependence of the unfolding are three residues with pKN = 3.3 and pKA = pKD = 4.4, one residue with pKN = pKA = 3.8 and pKD = 4.4, and one residue with pKN = 5.8 and pKA = pKD = 6.3, where A indicates the activated state. On the other hand, from the denaturant activity dependence of the shift factors required for making the master curves, the value of the intrinsic binding constant of the denaturant to the protein was found to be similar to that obtained from previous measurements at pH 5.5. Differences between the numbers of the binding sites of the denaturant on the denaturated and the native proteins, and between those on the activated and the native proteins were shown to be 5.3 and 2.1, respectively. The free energy of stabilization in the native-like environment also shows that the protein in the native state is more unstable than lysozyme.

Nucleotide binding to the chaperonin GroEL: non-cooperative binding of ATP analogs and ADP, and cooperative effect of ATP.

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by GroEL, which undergoes a large structural change by the ATP binding or hydrolysis. One of the main concerns of GroEL is the mechanism of the productive and cooperative structural change of GroEL induced by the nucleotide. We studied the cooperative nature of GroEL by nucleotide titration using isothermal titration calorimetry and fluorescence spectroscopy. Our results indicated that the binding of ADP and ATP analogs to a single ring mutant (SR1), as well as that to GroEL, was non-cooperative. Only ATP induces an apparently cooperative conformational change in both proteins. Furthermore, the fluorescence changes of pyrene-labeled GroEL indicated that GroEL has two kinds of nucleotide binding sites. The fluorescence titration result fits well with a model in which two kinds of binding sites are both non-cooperative and independent of each other. These results suggest that the binding and hydrolysis of ATP may be necessary for the cooperative transition of GroEL.

Absence of the thermal transition in apo-alpha-lactalbumin in the molten globule state. A study by differential scanning microcalorimetry.

To estimate the energy level of the molten globule state, the heat capacity function of apo-alpha-lactalbumin in the molten globule state has been examined using a scanning microcalorimeter at neutral pH. The results showed that the enthalpy difference between the molten globule state and presumed unfolded state by heating was almost zero at neutral pH, demonstrating that the molten globule state does not exhibit any co-operative transition upon heating. This is in agreement with the results already reported at acid pH, but is apparently in conflict with that recently reported with some assumptions at neutral pH.

Characterization of the critical state in protein folding. Effects of guanidine hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin.

The reversible unfolding and refolding kinetics of alpha-lactalbumin induced by concentration jump of guanidine hydrochloride were measured at pH 7.0 and 25 degrees C using tryptophan absorption at 292 nm, with varying concentrations of the denaturant and free Ca2+. The refolding reaction of alpha-lactalbumin from the fully unfolded (D) state occurs through the two stages: (1) instantaneous formation of a compact intermediate (the A state) that has a native-like secondary structure; (2) tight packing of the preformed secondary structure segments to lead finally to the native structure, this stage being the rate-determining step of the reaction and associated with acquisition of the specific structure necessary for strong Ca2+ binding. Under strongly native conditions, the observed kinetics of refolding is also complicated by the presence of a slow-folding species (10%) in the unfolded state. Considering these facts, the microscopic rate constants in folding and unfolding directions have been evaluated from the observed kinetics and from the equilibrium constants of the transitions among the native (N), A and D states. Close linear relationships have been found in the plots of the activation free energies, obtained from the microscopic rate constants, against the denaturant concentration. They are similar to the linear relationship between the free energy of unfolding and the denaturant concentration. It was demonstrated that the slope of the plots should be approximately proportional to a change in accessible surface area of the protein during the respective activation process, and that only a third of the difference in accessible surface area between A and N is buried in the critical activated state of folding. However, the selective effect of Ca2+ binding on the folding rate constant has been observed also, demonstrating that the specific Ca2+-binding substructure in the N state is already organized in the activated state. Thus, only a part of the protein molecule involving the Ca2+-binding region is organized in the activated state, with the other part of the molecule being left less organized, suggesting that the second stage of folding may be a sequential growing process of organized assemblage of the performed secondary structure segments.

The burst-phase intermediate in the refolding of beta-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy.

The kinetics of the guanidine hydrochloride-induced unfolding and refolding of bovine beta-lactoglobulin, a predominantly beta-sheet protein in the native state, have been studied by stopped-flow circular dichroism and absorption measurements at pH 3.2 and 4.5 degrees C. The refolding reaction was a complex process composed of different kinetic phases, while the unfolding was a single-phase reaction. Most notably, a burst-phase intermediate of refolding, which was formed during the dead time of stopped-flow measurements (approximately 18 ms), showed more intense ellipticity signals in the peptide region below 240 nm than the native state, yielding overshoot behavior in the refolding curves. We have investigated the spectral properties and structural stability of the burst-phase intermediate and also the structural properties in the unfolded state in 4.0 M guanidine hydrochloride of the protein and its disulfide-cleaved derivative. The main conclusions are: (1) the more intense ellipticity of the intermediate in the peptide region arises from formation of non-native alpha-helical structure in the intermediate, apparently suggesting that the folding of beta-lactoglobulin is not represented by a simple sequential mechanism. (2) The burst-phase intermediate has, however, a number of properties in common with the folding intermediates or with the molten globule states of other globular proteins whose folding reactions are known to be represented by the sequential model. These properties include: the presence of the secondary structure without the specific tertiary structure; formation of a hydrophobic core; broad unfolding transition of the intermediate; and rapidity of formation of the intermediate. The burst-phase intermediate of beta-lactoglobulin is thus classified as the same species as the molten globule state. (3) The circular dichroism spectra of beta-lactoglobulin and its disulfide-cleaved derivative in 4.0 M guanidine hydrochloride suggests the presence of the residual beta-structure in the unfolded state and the stabilization of the beta-structure by disulfide bonds. Thus; if this residual beta-structure is part of the native beta-structure and forms a folding initiation site, the folding reaction of beta-lactoglobulin may not necessarily be inconsistent with the sequential model. The non-native alpha-helices in the burst-phase intermediate may be formed in an immature part of the protein molecule because of the local alpha-helical propensity in this part.

Effect of GroEL on the re-folding kinetics of alpha-lactalbumin.

The effect of GroEL on the re-folding kinetics of apo- and holo-alpha-lactalbumin from the acidic molten globule state has been investigated by stopped-flow fluorescence measurements. GroEL retards the re-folding of apo-alpha-lactalbumin by interacting with the molten globule state of the protein. The binding constant was estimated to be in the order of 10(5) M-1 by analyzing the kinetic data quantitatively and was found to be much weaker than the binding between GroEL and disulfide-bond reduced alpha-lactalbumin, whose binding constant is in the order of 10(7) M-1. Our present results, together with the previous results, suggest that the state recognized by GroEL is not unique and that the binding strength varies with the state of a target protein. The binding between GroEL and the molten globule state of apo-alpha-lactalbumin becomes stronger with an increasing salt concentration; the binding constant is increased tenfold (from 10(5) to 10(6) M-1) by an increase in salt concentration from 0.05 to 0.25 M. The study of the effect of GroEL on the re-folding kinetics of holo-alpha-lactalbumin, which is represented by a bi-phasic process, shows that the slow phase is affected by GroEL in the same manner as observed in the apo-alpha-lactalbumin re-folding but that the fast phase is not affected by GroEL at all. This indicates that the binding rate of GroEL is faster than the slow phase but slower than the fast phase of the re-folding, and the bi-molecular rate constant of GroEL binding to the molten globule state of alpha-lactalbumin was estimated to be in the order of 10(6) M-1S-1.

Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES.

We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperonin GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin. The refolding reactions induced by stopped-flow pH jumps were monitored by alpha-lactalbumin tryptophan fluorescence. The simple single-exponential character of the free-refolding kinetics of the protein allowed us to quantitatively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for binding between GroEL and the refolding intermediate of alpha-lactalbumin by the non-linear least-squares method. When GroES was absent, the interaction between GroEL and alpha-lactalbumin could be well represented by a "cooperative-binding" model in which GroEL has two binding sites for alpha-lactalbumin with the affinity of the second site being tenfold weaker than that of the first, so that there is negative cooperativity between the two sites. The affinity between GroEL and alpha-lactalbumin was significantly reduced when ATP was present, while ADP and AMP-PNP did not alter the affinity. A comparison of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to the nucleotide-induced reduction of affinity. When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between GroEL and the refolding intermediate of alpha-lactalbumin. The affinity at a saturating concentration of ADP or AMP-PNP was about ten times lower than with GroEL alone. The ADP concentration at which the acceleration of the GroEL/ES-affected refolding of alphaLA was observed, was higher than the concentration at which the nucleotide-induced formation of the GroEL/ES complex took place. These results indicate that GroEL/ES complex formation itself is not enough to reduce the affinity for alpha-lactalbumin, and that further binding of the nucleotide to the GroEL/ES complex is required to reduce the affinity.

Exchange behavior of the H-bonded amide protons in the 3 to 13 helix of ribonuclease S.

The preceding article shows that there are eight highly protected amide protons in the S-peptide moiety of RNAase S at pH 5, 0 degrees C. The residues with protected NH protons are 7 to 13, whose amide protons are H-bonded in the 3 to 13 alpha-helix, and Asp 14, whose NH proton is H-bonded to the CO group of Val47. We describe here the exchange behavior of these eight protected protons as a function of pH. Exchange rates of the individual NH protons are measured by 1H nuclear magnetic resonance in D2O. A procedure is used for specifically labeling with 1H only these eight NH protons. The resonance assignments of the eight protons are made chiefly by partial exchange, through correlating the resonance intensities in spectra taken when the peptide is bound and when it is dissociated from S-protein in 3.5 M-urea-d4, in D2O, pH 2.3, -4 degrees C. The two remaining assignments are made and some other assignments are checked by measurements of the nuclear Overhauser effect between adjacent NH protons of the alpha-helix. There is a transition in exchange behavior between pH 3, where the helix is weakly protected against exchange, and pH 5 where the helix is much more stable. At pH 3.1, 20 degrees C, exchange rates are uniform within the helix within a factor of two, after correction for different intrinsic exchange rates. The degree of protection within the helix is only 10 to 20-fold at this pH. At pH 5.1, 20 degrees C, the helix is more stable by two orders of magnitude and exchange occurs preferentially from the N-terminal end. At both pH values the NH proton of Asp 14, which is just outside the helix, is less protected by an order of magnitude than the adjacent NH protons inside the helix. Opening of the helix can be observed below pH 3.7 by changes in chemical shifts of the NH protons in the helix. At pH 2.4 the changes are 25% of those expected for complete opening. Helix opening is a fast reaction on the n.m.r. time scale (tau much less than 1 ms) unlike the generalized unfolding of RNAase S which is a slow reaction. Dissociation of S-peptide from S-protein in native RNAase S at pH 3.0 also is a slow reaction. Opening of the helix below pH 3.7 is a two-state reaction, as judged by comparing chemical shifts with exchange rates. The exchange rates at pH 3.1 are predicted correctly from the changes in chemical shift by assuming that helix opening is a two-state reaction. At pH values above 3.7, the nature of the helix opening reaction changes. These results indicate that at least one partially unfolded state of RNAase S is populated in the low pH unfolding transition.

Nature and locations of the most slowly exchanging peptide NH protons in residues 1 to 19 of ribonuclease S.

The locations have been found of the eight most slowly exchanging peptide protons in residues 1 to 19 of ribonuclease S. The resonance lines of these eight protons are resolved by proton magnetic resonance at 360 MHz when either S-peptide (residues 1 to 19) or peptide 1-15 is bound to S-protein (residues 21 to 124). Other peptide protons have been removed by exchange in the sample preparation [( 1H]S-peptide is added to deuterated S-protein in D2O), and also by exchange-out of the less protected protons in residues 1 to 19. At pH 5.1, 0 degrees C, there is a 100-fold difference in rates of exchange between the eight most protected protons and the less protected protons of S-peptide. The highly protected protons are protected 10(4)-fold compared to free S-peptide. The protected protons have been identified by 1H nuclear magnetic resonance after denaturing ribonuclease S in greater than or equal to 3 M-urea-d4, D2O, pH 2.3, -4 degrees C, followed by comparing the chemical shifts of the remaining eight protons with the known -NH spectrum of the free peptide, which has been assigned from the two-dimensional homonuclear correlated spectrum and by comparison with earlier work. The eight highly protected NH protons are localized in one segment, residues 7 to 14. All eight protons are H-bonded: those of residues 7 to 13 are H-bonded within the 3-13 alpha-helix and that of residue 14 is H-bonded to the beta-sheet. The NH proton of residue 16, which also is H-bonded to the beta-sheet, is not one of the highly protected protons. Both the N atoms of the eight NH groups and also the O atoms of their CO acceptor groups are shielded from solvent in most cases, according to the molecular area calculations of Finney (1978).

Nucleotide-induced transition of GroEL from the high-affinity to the low-affinity state for a target protein: effects of ATP and ADP on the GroEL-affected refolding of alpha-lactalbumin.

We studied the refolding kinetics of alpha-lactalbumin in the presence of wild-type GroEL and its ATPase-deficient mutant D398A at various concentrations of nucleotides (ATP and ADP). We evaluated the apparent binding constant between GroEL and the alpha-lactalbumin refolding intermediate quantitatively by numerical simulation analysis of the alpha-lactalbumin refolding curves in the presence and absence of GroEL. The binding constant showed a co-operative decrease with an increase in ATP concentration, whereas the binding constant decreased in a non-co-operative manner with respect to ADP concentration. For the D398A mutant, the ATP-induced decrease in affinity occurred much faster than the steady-state ATP hydrolysis by this mutant, suggesting that ATP binding to GroEL rather than ATP hydrolysis, was responsible for the co-operative decrease in the affinity for the target protein. We thus analyzed the nucleotide-concentration dependence of affinity of GroEL for the target protein using an allosteric Monod-Wyman-Changeux model in which GroEL underwent an ATP-induced co-operative conformational transition between the high-affinity and low-affinity states of the target protein. The transition midpoint of the ATP-induced transition of GroEL has been found to be around 30 microM, in good agreement with the midpoint evaluated in other structural studies of GroEL. The results show that the observed difference between ATP and ADP-induced transitions of GroEL are brought about by a small difference in an allosteric parameter (the ratio of the nucleotide affinities of GroEL in the high-affinity and the low-affinity states), i.e. 4.1 for ATP and 2.6 for ADP.

Fast folding of Escherichia coli cyclophilin A: a hypothesis of a unique hydrophobic core with a phenylalanine cluster.

Escherichia coli cyclophilin A, a 164 residue globular protein, shows fast and slow phases of refolding kinetics from the urea-induced unfolded state at pH 7.0. Given that the slow phases are independent of the denaturant concentration and may be rate-limited by cis/trans isomerizations of prolyl peptide bonds, the fast phase represents the true folding reaction. The extrapolation of the fast-phase rate constant to 0 M urea indicates that the folding reaction of cyclophilin A is extraordinarily fast and has about 700 s(-1) of the rate constant. Interrupted refolding experiments showed that the protein molecules formed in the fast phase had already been fully folded to the native state. This finding overthrows the accepted view that the fast folding is observed only in small proteins of fewer than 100 amino acid residues. Examination of the X-ray structure of cyclophilin A has shown that this protein has only one unique hydrophobic core (phenylalanine cluster) formed by evolutionarily conserved phenylalanine residues, and suggests that this architecture of the molecule may be responsible for the fast folding behavior.

Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL.

We have analyzed the effect of the chaperonin GroEL on the refolding kinetics of staphylococcal nuclease and its three mutants by stopped-flow fluorescence measurements. It was found that a transient folding intermediate of staphylococcal nuclease was tightly bound to GroEL and refolded in the GroEL-bound state without releasing the non-native protein in solution, and the refolding rate in the GroEL-bound state was 0.01 s-1. The GroEL-affected refolding of the nuclease appears to be in decided contrast to that of apo-alpha-lactalbumin reported in our previous study, wherein alpha-lactalbumin was shown to be more weakly bound by GroEL and to refold in the free state in solution. In spite of the apparent difference between the proteins, the GroEL-affected refolding reactions of both the proteins can be represented by a common unified reaction scheme. On the basis of this scheme, the binding constant between the nuclease intermediate and GroEL was estimated to be larger than 10(9) M-1. The stoichiometry of binding of the nuclease and its mutants to GroEL was found to be two (nuclease/GroEL 14-mer). The increase in ionic strength resulted in a weakening of the interaction between the nuclease and GroEL, which was attributed to a weakening of the electrostatic attraction between the two proteins as a result of electrostatic screening by ions. Although ATP was found to accelerate the GroEL-affected refolding of the nuclease, the refolding rate was still far from the rate of the free refolding. The free refolding behavior of the nuclease and its mutants was restored in the presence of the cochaperonin GroES and ATP.

Dominant forces in the recognition of a transient folding intermediate of alpha-lactalbumin by GroEL.

GroEL is known to retard the refolding of apo-alpha-lactalbumin by interacting with the molten globule state of the protein. In order to investigate the dominant forces in this interaction, the GroEL-affected kinetic refolding of apo-alpha-lactalbumin from its acidic molten globule state was studied at different temperatures and in the presence of different kinds of monovalent cations at a fixed temperature (25 degrees C), by stopped-flow fluorescence measurements. The binding constant between GroEL and alpha-lactalbumin in the molten globule state was evaluated quantitatively from the kinetic refolding curves in the absence and presence of GroEL. The binding was found to be entropy-driven at room temperature and the heat capacity change for the binding was found to be largely negative (-3.6 kJ mol-1.K-1), indicating that GroEL binds to alpha-lactalbumin through hydrophobic interactions. The study of the effect of different monovalent cations at various ionic strengths shows that the binding is strengthened by electrostatic screening by ions, demonstrating the importance of electrostatic interactions. The relationship of these results with a putative target recognition site of GroEL will be discussed.

Equilibrium and kinetics of the folding of equine lysozyme studied by circular dichroism spectroscopy.

The equilibrium unfolding and the kinetics of unfolding and refolding of equine lysozyme, a Ca2+-binding protein, were studied by means of circular dichroism spectra in the far and near-ultraviolet regions. The transition curves of the guanidine hydrochloride-induced unfolding measured at 230 nm and 292.5 nm, and for the apo and holo forms of the protein have shown that the unfolding is well represented by a three-state mechanism in which the molten globule state is populated as a stable intermediate. The molten globule state of this protein is more stable and more native-like than that of alpha-lactalbumin, a homologous protein of equine lysozyme. The kinetic unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by stopped-flow circular dichroism. The observed unfolding and refolding curves both agreed well with a single-exponential function. However, in the kinetic refolding reactions below 3 M guanidine hydrochloride, a burst-phase change in the circular dichroism was present, and the burst-phase intermediate in the kinetic refolding is shown to be identical with the molten globule state observed in the equilibrium unfolding. Under a strongly native condition, virtually all the molecules of equine lysozyme transform the structure from the unfolded state into the molten globule, and the subsequent refolding takes place from the molten globule state. The transition state of folding, which may exist between the molten globule and the native states, was characterized by investigating the guanidine hydrochloride concentration-dependence of the rate constants of refolding and unfolding. More than 80% of the hydrophobic surface of the protein is buried in the transition state, so that it is much closer to the native state than to the molten globule in which only 36% of the surface is buried in the interior of the molecule. It is concluded that all the present results are best explained by a sequential model of protein folding, in which the molten globule state is an obligatory folding intermediate on the pathway of folding.

Kinetic refolding of beta-lactoglobulin. Studies by synchrotron X-ray scattering, and circular dichroism, absorption and fluorescence spectroscopy.

beta-Lactoglobulin (beta LG) is a predominantly beta-sheet protein with a markedly high helical propensity and forms non-native alpha-helical intermediate in the refolding process. We measured the refolding reaction of beta LG with various techniques and characterized the folding kinetics and the structure of the intermediate formed within the burst phase of measurements, i.e. the burst-phase intermediate. Time-resolved stopped-flow X-ray scattering measurements using the integral intensity of scattering show that beta LG forms a compact, globular structure within 30 ms of refolding. The averaged radius of gyration within 100 ms is only 1.1 times larger than that in the native state, ensuring that the burst-phase intermediate is compact. The presence of a maximum peak in a Kratky plot shows a globular shape attained within 100 ms of refolding. Stopped-flow circular dichroism, tryptophan absorption and fluorescence spectroscopy show that pronounced secondary structure regains rapidly in the burst phase with concurrent non-native alpha-helix formation, and that the subsequent compaction process is accompanied by annealing of non-native secondary structure and slow acquisition of tertiary structure. These findings strongly suggest that both compaction and secondary structure formation in protein folding are quite rapid processes, taking place within a millisecond time-scale. The structure of the burst-phase intermediate in beta LG refolding was characterized as having a compact size, a globular shape, a hydrophobic core, substantial beta-sheets and remarkable non-native alpha-helical structure, but little tertiary structure. These results suggest that both local interactions and non-local hydrophobic interactions are dominant forces early in protein folding. The interplay of local and non-local interactions throughout folding processes is important in understanding the mechanisms of protein folding.