Thermal denaturation of spinach plastocyanin: effect of copper site oxidation state and molecular oxygen

The thermal denaturation of the cupredoxin plastocyanin (PC) from spinach has been studied with the aim of improving the understanding of factors involved in the conformational stability of antiparallel beta-sheet proteins. Studies using differential scanning calorimetry have been complemented with nuclear magnetic resonance spectroscopy, absorbance spectroscopy, dynamic light scattering, and mass spectrometry in elucidation of the effect of the copper-site oxidation state on the irreversible thermal denaturation process. Our results indicate that copper-catalyzed oxidation of the metal-ligating cysteine is the sole factor resulting in thermal irreversibility. However, this can be prevented in reduced protein by the removal of molecular oxygen. Application of a two-state equilibrium transition model to the folding process thus allowed the extraction of thermodynamic parameters for the reduced protein (Delta(trs)H = 494 kJ mol(-1), DeltaH(vH) = 343 kJ mol(-1), and T(m) = 71 degrees C). However, anaerobically denatured oxidized protein and all aerobically denatured species undergo covalent modification as a result of the copper-catalyzed oxidation of the metal-ligating cysteine residue resulting in the formation of both oxidized monomers and disulfide-linked dimers. On the basis of these results, a general mechanism for the irreversible thermal denaturation of cupredoxins is proposed. The results presented here also indicate that PC, as opposed to the previously characterized homologous protein azurin, unfolds via at least one significantly populated intermediate state (DeltaH(vH)/Delta(trs)H = 0.7) despite the almost identical native state topologies of these proteins. These findings will aid the characterization of the stability of PC and other cupredoxins and possibly of all cysteine-ligating metal-binding proteins.     

Multi-State Proteins: Approach Allowing Experimental Determination of the Formation Order of Structure Elements in the Green Fluorescent Protein

The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named μ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are “tested” by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an μ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.     

Transition state of the rate-limiting step of heat denaturation of Cry3A delta-endotoxin

Heat denaturation of Cry3A delta-endotoxin from Bacillus thuringiensis var. tenebrionis and its 55 kDa fragment was studied by differential scanning microcalorimetry at low pH. Analysis of the calorimetric data has shown that denaturation of Cry3A delta-endotoxin is a nonequilibrium process at heating rates from 0. 125 to 2 K/min. This means that the stability of delta-endotoxin (the apparent temperature of denaturation Tm) under these conditions is under kinetic control rather than under thermodynamic control. It has been shown that heat denaturation of this protein is a one-step kinetic process. The enthalpy of the process and its activation energy were measured as functions of temperature. The data obtained allow confirmation of the fact that the conformation of delta-endotoxin at the transition state only slightly differs from its native conformation with respect to compactness and extent of hydration. The comparison of the activation energy for intact delta-endotoxin and the 55 kDa fragment showed that the transition of the molecule to a transition state does not cause any changes in the conformation of three N-terminal alpha-helices. Complete removal of the N-terminal domain of delta-endotoxin and 40 amino acids from the C-terminus beta-sheet domain III causes an irreversible loss of the tertiary structure. Thus, during protein folding the nucleation core determining protein stability does not involve its three initial alpha-helices but may include the remaining alpha-helices of the N-terminal domain. The functional significance of peculiarities of structure arrangement of the delta-endotoxin molecule is discussed.     

Stable intermediate states and high energy barriers in the unfolding of GFP

We present a study of the denaturation of a truncated, cycle3 variant of green fluorescent protein (GFP). Chemical denaturation is used to unfold the protein, with changes in structure being monitored by the green fluorescence, tyrosine fluorescence and far-UV circular dichroism. The results show that the denaturation behaviour of GFP is complex compared to many small proteins: equilibrium is established only very slowly, over the time course of weeks, suggesting that there are high folding/unfolding energy barriers. Unfolding kinetics confirm that the rates of unfolding at low concentrations of denaturant are very low, consistent with the slow establishment of the equilibrium. In addition, we find that GFP significantly populates an intermediate state under equilibrium conditions, which is compact and stable with respect to the unfolded state (m(IU)=4.6 kcal mol(-1) M(-1) and Delta G(IU)=12.5 kcal mol(-1)). The global and local stability of GFP was probed further by measuring the hydrogen/deuterium (H/D) NMR exchange rates of more than 157 assigned amide protons. Analysis at two different values of pH showed that amide protons within the beta-barrel structure exchange at the EX2 limit, consequently, free energies of exchange could be calculated and compared to those obtained from the denaturation-curve studies providing further support for the three-state model and the existence of a stable intermediate state. Analysis reveals that amide protons in beta-strands 7, 8, 9 and 10 have, on average, higher exchange rates than others in the beta-barrel, suggesting that there is greater flexibility in this region of the protein. Forty or so amide protons were found which do not undergo significant exchange even after several months and these are clustered into a core region encompassing most of the beta-strands, at least at one end of the barrel structure. It is likely that these residues play an important role in stabilizing the structure of the intermediate state. The intermediate state observed in the chemical denaturation studies described here, is similar to that observed at pH 4 in other studies.     

Residual ordered structure in denatured proteins and the problem of protein folding

Structural characteristics of numerous globular proteins in the denatured state have been reviewed using literature data. Recent more precise experiments show that in contrast to the conventional standpoint, proteins under strongly denaturing conditions do not unfold completely and adopt a random coil state, but contain significant residual ordered structure. These results cast doubt on the basis of the conventional approach representing the process of protein folding as a spontaneous transition of a polypeptide chain from the random coil state to the unique globular structure. The denaturation of proteins is explained in terms of the physical properties of proteins such as stability, conformational change, elasticity, irreversible denaturation, etc. The spontaneous renaturation of some denatured proteins most probably is merely the manifestation of the physical properties (e.g., the elasticity) of the proteins per se, caused by the residual structure present in the denatured state. The pieces of the ordered structure might be the centers of the initiation of renaturation, where the restoration of the initial native conformation of denatured proteins begins. Studies on the denaturation of proteins hardly clarify how the proteins fold into the native conformation during the successive residue-by-residue elongation of the polypeptide chain on the ribosome.     

Folding and aggregation are selectively influenced by the conformational preferences of the alpha-helices of muscle acylphosphatase

The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.     

Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues.

A model is proposed to account for the observation that the denaturation of small proteins apparently occurs in two kinetic phases. It is suggested that only one of these phases--the fast one--is actually an unfolding process. The slow phase is assumed to arise from the cis-trans isomerism of proline residues in the denaturated protein. From model compound data, it is shown that the expected rate for isomerism is in satisfactory agreement with the rates actually observed for protein folding. It is also shown that a simple model of protein unfolding based on the isomerism concept is very successful in accounting for many known experimental characteristics of the kinetics and thermodynamic of protein denaturation. Thus, the model is able to predict that two kinetic phases will be seen in the transition region while none are seen in the base-line regions, that both the fast and slow refolding phases lead to the native protein as the product, that the fast phase becomes the only observable phase for jumps ending far in the denatured base-line region, that most or all small proteins show a limiting low-temperature activation energy of ca. 20,000 cal, and that the relaxtion time for the slow phase seen in cytochrome c denaturation is much shorter than for all other small proteins. By utilizing "double-jump" experiments, it is shown directly that the slow phase is not part of the unfolding process but that it corresponds to a transition among two or more denatured forms which have identical spectroscopic (286.5 nm) properties. Thus, the slow relaxation is "invisible" except in the transition region where it couples to the fast unfolding equilibrium. Finally, since the present model assumes that only one of the major kinetic phases seen in denaturation reactions is concerned with the denaturation process per se, it is in agreement with numerous thermodynamic studies which show consistency with the two-state model for unfolding.     

An approach for the assessment of the order of disruption of the elements of protein structure upon protein unfolding: A study of carbonic anhydrase B

An experimental approach named μ-analysis has been developed in order to elucidate the sequence of the loss of ordered structure by elements of a protein during the denaturation of the molecule. This approach is applicable for the analysis of proteins that fold (unfold) in a multistep process that involve the formation (destruction) of a range of intermediate states. The concept of the approach consists in systematic analysis of mutagenized forms of the protein with point substitutions of hydrophobic amino-acid residues and additional cysteine bridges. Importantly, the substitutions of the amino-acid residues must be localized to the same structural elements of the protein. Point substitutions of hydrophobic amino-acid residues mainly provide information on the structural elements of the protein that are disrupted at the final stages of protein denaturation. The addition of cysteine bridges to the surface of the protein molecule allows investigation of structural elements of the protein that are the first to unfold upon protein denaturation. Calorimetric studies of non-equilibrium melting of bovine carbonic anhydrase B yielded information on the rate constants of the unfolding of ten mutant forms of the protein. The analysis of the effects of mutations on the rates of different stages of protein unfolding allowed for elucidation of the order of disruption of structural elements of carbonic anhydrase B upon thermal denaturation.     

Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations

The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).     

Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (>or=35 degrees C), tailspike refolding yields were increased significantly in the presence of 1-4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.     

The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.     

A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies

Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state or time-resolved fluorescence resonance energy transfer (FRET) detection of the kinetics of protein folding require relatively large quantities (approximately 10-100 mg) of site-specific doubly labeled protein samples. Engineered cysteine residues are common targets for labeling of proteins. The challenge here is to develop methods for selective modification of one of two reactive sulfhydryl groups in a protein molecule. A general systematic procedure for selective labeling of each of two cysteine residues in a protein molecule was developed, using Escherichia coli adenylate kinase (AKe) as a model protein. Potential sites for insertion of cysteine residues were selected by examination of the crystal structure of the protein. A series of single-cysteine mutants was prepared, and the rates of the reaction of each engineered cysteine residue with a reference reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] were determined. Two-cysteine mutants were prepared by selection of pairs of sites for which the ratio of this reaction rate constant was high (>80). The conditions for the selective labeling reaction were optimized. In a first cycle of labeling, the more reactive cysteine residue was labeled with a fluorescent probe (donor). The second probe was attached to the less reactive site under unfolding conditions in the second cycle of labeling. The doubly and singly labeled mutants retained full enzymatic activity and the capacity for a reversible folding-unfolding transition. High yields (70-90%) of the preparation of the pure, site-specific doubly labeled AK mutant were obtained. The procedure described herein is a general outline of procedures, which can meet the double challenge of both site specificity and large-scale preparation of doubly labeled proteins.     

Mapping the folding pathway of an immunoglobulin domain: structural detail from Phi value analysis and movement of the transition state

BACKGROUND: Do proteins that have the same structure fold by the same pathway even when they are unrelated in sequence? To address this question, we are comparing the folding of a number of different immunoglobulin-like proteins. Here, we present a detailed protein engineering phi value analysis of the folding pathway of TI I27, an immunoglobulin domain from human cardiac titin. RESULTS: TI I27 folds rapidly via a kinetic intermediate that is destabilized by most mutations. The transition state for folding is remarkably native-like in terms of solvent accessibility. We use phi value analysis to map this transition state and show that it is highly structured; only a few residues close to the N-terminal region of the protein remain completely unfolded. Interestingly, most mutations cause the transition state to become less native-like. This anti-Hammond behavior can be used as a novel means of obtaining additional structural information about the transition state. CONCLUSIONS: The residues that are involved in nucleating the folding of TI I27 are structurally equivalent to the residues that form the folding nucleus in an evolutionary unrelated fibronectin type III protein. These residues form part of the common structural core of Ig-like domains. The data support the hypothesis that interactions essential for defining the structure of these beta sandwich proteins are also important in nucleation of folding.     

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.     

Acid denaturation and refolding of green fluorescent protein

Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.     

A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.     

A pH dependence study on the unfolding and refolding of apoazurin

Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a β-sheet protein arranged into a single domain. Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process. The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains. However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be −27.8±2.4 kJ mol⁻¹. From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1±6.9 kJ/mol and 12.9±8.1 kJ/mol, respectively. It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity—in particular, from the presence of Zn(II) azurin. We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5. The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH. We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains. When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion. Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.     

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.