Folding of the multidomain ribosomal protein L9: the two domains fold independently with remarkably different rates

The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy. One-dimensional 1H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (Tm = 67 degrees C for the C-terminal domain, 80 degrees C for the N-terminal domain). NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain. At 72 degrees C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1. For the C-terminal domain, saturation transfer experiments performed at 69 degrees C were used to determine the folding rate constant, 3.3 s-1, and the unfolding rate constant, 9.0 s-1. Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain. The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1, respectively, for the N-terminal domain at 25 degrees C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain. The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding. The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein. The remarkably different rate constants between the N- and C-terminal domains suggest that the two domains can fold and unfold independently. The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important.     

Site-specific experiments on folding/unfolding of Jun coiled coils: thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine-18

The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 alpha-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil dimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate constants for GCN4 as determined by chevron analysis and NMR in differing temperature regimes.     

Kinetic analysis of folding and unfolding the 56 amino acid IgG-binding domain of streptococcal protein G.

The 56 amino acid B domain of protein G (GB) is a stable globular folding unit with no disulfide cross-links. The physical properties of GB offer extraordinary flexibility for evaluating the energetics of the folding reaction. The protein is monomeric and very soluble in both folded and unfolded forms. The folding reaction has been previously examined by differential scanning calorimetry (Alexander et al., 1992) and found to exhibit two-state unfolding behavior over a wide pH range with an unfolding transition near 90 degrees C (GB1) at neutral pH. Here, the kinetics of folding and unfolding two naturally occurring versions of GB have been measured using stopped-flow mixing methods and analyzed according to transition-state theory. GB contains no prolines, and the kinetics of folding and unfolding can be fit to a single, first-order rate constant over the temperature range of 5-35 degrees C. The major thermodynamic changes going from the unfolded state to the transition state are (1) a large decrease in heat capacity (delta Cp), indicating that the transition state is compact and solvent inaccessible relative to the unfolded state; (2) a large loss of entropy; and (3) a small increase in enthalpy. The most surprising feature of the folding of GB compared to that of previously studied proteins is that its folding approximates a rapid diffusion controlled process with little increase in enthalpy going from the unfolded to the transition state.     

Transient intermediary states with high and low folding probabilities in the apparent two-state folding equilibrium of ACBP at low pH

Measurements of the stability as a function of pH for the acyl-coenzyme A binding protein (ACBP) has shown a significant difference in the pH transition midpoint measured by NMR spectroscopy at pH 3.12 and the transition midpoint measured at pH 2.92 and 2.97 by circular dichroism and by fluorescence spectroscopy, respectively. A similar behavior has not been observed in other proteins. It is suggested that these differences arise because the population of the unfolded molecules still contains significant amounts of native like secondary and tertiary structure. NMR spectroscopy measures the concentration of the two components of the folding unfolding equilibrium individually, whereas circular dichroism and fluorescence measure the concentration of the conformations of the light-absorbing chromophores present in both the folded and the unfolded molecules. In the narrow pH range, nascent structure can be detected as the average amount of secondary structure per unfolded molecule and hydrophobic interactions in the population of unfolded molecules. These structures are not observable immediately by NMR spectroscopy; however, a chemical shift analysis of the peptide backbone (13)C chemical shift indicates strongly the existence of short-lived and transient helical structures at pH 2.3. Magnetization transfer studies have been applied to study the equilibrium between folded and unfolded ACBP near the pH transition point measured by NMR. This study has shown that there are two categories of subpopulations in the population of unfolded ACBP. One for which magnetization can be transferred to the folded form during the folding process, and one for which transfer is not observed. The molecules of the latter population of unfolded protein apparently, do not fold within the time-frame of the magnetization transfer experiment. This result suggests the existence of a subpopulation of the acid-unfolded protein molecules with a high propensity for folding. It is suggested that in this subpopulation, a particular set of native like interactions in the peptide backbone and between side-chains in the peptide chain have to be formed.     

Test for cooperativity in the early kinetic intermediate in lysozyme folding

During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.     

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions

Plastocyanin is a predominantly beta-sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo-plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three-dimensional triple-resonance NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The (3)J(HN,Halpha) coupling constants, (15)N-edited proton-proton nuclear Overhauser effects (NOEs), and (15)N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to beta-strands in the folded protein exhibit small upfield shifts of the (13)C(alpha) and (13)CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the beta region of (phi,psi) space. This is further supported by the presence of strong sequential d(alphaN)(i, i + 1) NOEs throughout the sequence. The few d(NN)(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the (13)C(alpha), (13)CO, and (1)H(alpha) resonances relative to random coil values and by the decreased values of the (3)J(HN,Halpha) coupling constants. The (15)N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form beta-strands in the native protein spontaneously populate the beta-region of (phi,psi) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo-plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.     

Folding kinetics and thermodynamics of Pseudomonas syringae effector protein AvrPto provide insight into translocation via the type III secretion system

In order to infect their hosts, many Gram-negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a well-studied effector protein of Pseudomonas syringae pv tomato. The presence of a readily observed unfolded population of AvrPto in aqueous solution and the lack of a known secretion chaperone make it ideal for studying the kinetic and thermodynamic characteristics that facilitate translocation. Application of Nzz exchange spectroscopy revealed a global, two-state folding equilibrium with 16% unfolded population, a folding rate of 1.8 s(-1), and an unfolding rate of 0.33 s(-1) at pH 6.1. TrAvrPto stability increases with increasing pH, with only 2% unfolded population observed at pH 7.0. The R(1) relaxation of TrAvrPto, which is sensitive to both the global anisotropy of folded TrAvrPto and slow exchange between folded and unfolded conformations, provided independent verification of the global kinetic rate constants. Given the acidic apoplast in which the pathogen resides and the more basic host cytoplasm, these results offer an intriguing mechanism by which the pH dependence of stability and slow folding kinetics of AvrPto would allow efficient translocation of the unfolded form through the TTSS and refolding into its functional folded form once inside the host.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force.

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.     

Pressure-Temperature Analysis of the Stability of the CTL9 Domain Reveals Hidden Intermediates

The observation of two-state unfolding for many small single-domain proteins by denaturants has led to speculation that protein sequences may have evolved to limit the population of partially folded states that could be detrimental to fitness. How such strong cooperativity arises from a multitude of individual interactions is not well understood. Here, we investigate the stability and folding cooperativity of the C-terminal domain of the ribosomal protein L9 in the pressure-temperature plane using site-specific NMR. In contrast to apparent cooperative unfolding detected with denaturant-induced and thermal-induced unfolding experiments and stopped-flow refolding studies at ambient pressure, NMR-detected pressure unfolding revealed significant deviation from two-state behavior, with a core region that was selectively destabilized by increasing temperature. Comparison of pressure-dependent NMR signals from both the folded and unfolded states revealed the population of at least one invisible excited state at atmospheric pressure. The core destabilizing cavity-creating I98A mutation apparently increased the cooperativity of the loss of folded-state peak intensity while also increasing the population of this invisible excited state present at atmospheric pressure. These observations highlight how local stability is subtly modulated by sequence to tune protein conformational landscapes and illustrate the ability of pressure- and temperature-dependent studies to reveal otherwise hidden states.     

Unfolding kinetics of dimeric creatine kinase measured by stopped-flow small angle X-ray scattering

The unfolding kinetics of creatine kinase (CK) in various concentrations of urea or guanidine hydrochloride (GuHCl) was investigated by small angle X-ray scattering (SAXS) using synchrotron radiation, and compared with the results obtained by stopped-flow circular dichroism and stopped-flow fluorescence. Using the three methods, the unfolding kinetics of CK fits well to a single exponential function with similar apparent rate constants, and the amplitude of the monophasic kinetics covers the entire range of the equilibrium values. The results suggest that the unfolding time-course measured by integrated SAXS intensity corresponds to the intramolecular loss of globular structure. The refolding kinetics of 8 M urea-denatured CK was monitored in a stopped-flow apparatus by following the spectroscopic changes, and the final state of folding was investigated by SAXS. A substantial part of the ellipticity is recovered within a burst phase, indicating that the secondary structure forms at an early stage in refolding. The R(g) value of the final folded state was 33.6 A when the folding buffer contained 20% glycerol, which is characteristic of native-like compactness and globularity.     

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.     

Determination of thermodynamics and kinetics of RNA reactions by force

Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.     

Replica exchange simulation of reversible folding/unfolding of the Trp-cage miniprotein in explicit solvent: on the structure and possible role of internal water

We simulate the folding/unfolding equilibrium of the 20-residue miniprotein Trp-cage. We use replica exchange molecular dynamics simulations of the AMBER94 atomic detail model of the protein explicitly solvated by water, starting from a completely unfolded configuration. We employ a total of 40 replicas, covering the temperature range between 280 and 538 K. Individual simulation lengths of 100 ns sum up to a total simulation time of about 4 micros. Without any bias, we observe the folding of the protein into the native state with an unfolding-transition temperature of about 440 K. The native state is characterized by a distribution of root mean square distances (RMSD) from the NMR data that peaks at 1.8A, and is as low as 0.4A. We show that equilibration times of about 40 ns are required to yield convergence. A folded configuration in the entire extended ensemble is found to have a lifetime of about 31 ns. In a clamp-like motion, the Trp-cage opens up during thermal denaturation. In line with fluorescence quenching experiments, the Trp-residue sidechain gets hydrated when the protein opens up, roughly doubling the number of water molecules in the first solvation shell. We find the helical propensity of the helical domain of Trp-cage rather well preserved even at very high temperatures. In the folded state, we can identify states with one and two buried internal water molecules interconnecting parts of the Trp-cage molecule by hydrogen bonds. The loss of hydrogen bonds of these buried water molecules in the folded state with increasing temperature is likely to destabilize the folded state at elevated temperatures.     

Preorganized secondary structure as an important determinant of fast protein folding

The folding and unfolding kinetics of the B-domain of staphylococcal protein A, a small three-helix bundle protein, were probed by NMR. The lineshape of a single histidine resonance was fit as a function of denaturant to give folding and unfolding rate constants. The B-domain folds extremely rapidly in a two-state manner, with a folding rate constant of 120,000 s-1, making it one of the fastest-folding proteins known. Diffusion-collision theory predicts folding and unfolding rate constants that are in good agreement with the experimental values. The apparent rate constant as a function of denaturant ('chevron plot') is predicted within an order of magnitude. Our results are consistent with a model whereby fast-folding proteins utilize a diffusion-collision mechanism, with the preorganization of one or more elements of secondary structure in the unfolded protein.     

Kinetics of folding and unfolding of alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin.

Stopped flow CD (SFCD) kinetic studies of self-assembly of coiled coils of rabbit alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin (NPTm) are reported. The protein was denatured in 6 M urea buffer, then renatured by 10-fold dilution into benign saline buffer. Folding was monitored by SFCD in the backbone region (222 nm). Protein chains are shown to be totally unfolded (and separated in the reduced species) in the initial denaturing medium and fully folded as two-chain coiled coils in the final benign medium. In all cases of folding in benign buffer of totally unfolded chains, two phases were found in the folding process: a fast phase (less than 0.04 s, the SFCD dead time), in which an intermediate state with about 70% of the equilibrium ellipticity forms; followed by a slower, observable phase that completes the folding. The slow phase is first order (k-1 = 1.6 s at 20 degrees C), signifying that chain association for reduced samples occurs in the fast phase. In contrast, folding in benign buffer from an initial state with 70% of the equilibrium ellipticity is all fast, suggesting that the folding intermediate is not an equilibrium species. Cross-linking at Cys-190 increases the helix content of the fast-formed intermediate state to about 85% of the equilibrium value, but leaves the rate constant of the slow phase unchanged. In NPTm, which does not form high aggregates at low ionic strength, the rate of the observable phase is almost independent of ionic strength in the range of approximately 0.15-0.6 M, but is reduced one to two orders of magnitude by further reduction to 0.026 M. In folding from totally unfolded chains, the rate is reduced less than one order of magnitude by changing the final state to about 50% folded. In contrast to folding, unfolding of alpha alpha-tropomyosin from the native state is all fast.     

Unfolding rates of barstar determined in native and low denaturant conditions indicate the presence of intermediates

The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of folding from urea and from GdnHCl unfolded forms are extrapolated linearly with denaturant concentration, the same rate is obtained for folding in zero denaturant. Similar linear extrapolations of rates of unfolding in urea and GdnHCl yield, however, different unfolding rates in zero denaturant, indicating that such linear extrapolations are not valid. It has been difficult, for any protein, to determine unfolding rates under nativelike conditions in direct kinetic experiments. Using a novel strategy of coupling the reactivity of a buried cysteine residue with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the unfolding reaction of barstar, the global unfolding and refolding rates have now been determined in low denaturant concentrations. The logarithms of unfolding rates obtained at low urea and GdnHCl concentrations show a markedly nonlinear dependence on denaturant concentration and converge to the same unfolding rate in the absence of denaturant. It is shown that the native protein can sample the fully unfolded conformation even in the absence of denaturant. The observed nonlinear dependences of the logarithms of the refolding and unfolding rates observed for both denaturants are shown to be due to the presence of (un)folding intermediates and not due to movements in the position of the transition state with a change in denaturant concentration.     

Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

Transition state in the folding of alpha-lactalbumin probed by the 6-120 disulfide bond.

The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of alpha-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact alpha-lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate-controlled folding model and the multiple-pathway folding model, that we had proposed previously. All of the data supported the multiple-pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of alpha-lactalbumin is not an obligatory intermediate. The cleavage of the 6-120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6-120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of alpha-lactalbumin is discussed.