Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane

In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.     

Unfolding study of a trimeric membrane protein AcrB

The folding of a multi‐domain trimeric α‐helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two‐state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter‐subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate‐limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrB_Δloop, from the SDS unfolded state. The CD spectrum of the refolded AcrB_Δloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re‐association of the trimer might be the limiting factor to obtain folded wild‐type AcrB.     

Disulfide formation and stability of a cysteine-rich repeat protein from Helicobacter pylori

Helicobacter pylori cysteine-rich proteins (Hcps) are disulfide-containing repeat proteins. The repeating unit is a 36-residue, disulfide-bridged, helix-loop-helix motif. We use the protein HcpB, which has four repeats and four disulfide bridges arrayed in tandem, as a model to determine the thermodynamic stability of a disulfide-rich repeat protein and to study the formation and the contribution to stability of the disulfide bonds. When the disulfide bonds are intact, the chemical unfolding of HcpB at pH 5 is cooperative and can be described by a two-state reaction. Thermal unfolding is reversible between pH 2 and 5 and irreversible at higher pH 5. Differential scanning calorimetry shows noncooperative structural changes preceding the main thermal unfolding transition. Unfolding of the oxidized protein is not an all-or-none two-state process, and the disulfide bonds prevent complete unfolding of the polypeptide chain. The reduced protein is significantly less stable and does not unfold in a cooperative way. During oxidative refolding of the fully reduced protein, all the possible disulfide intermediates with a correct disulfide bond are formed. Formation of "wrong" (non-native) disulfide bonds could not be demonstrated, indicating that the reduced protein already has some partial repeating structure. There is a major folding intermediate with disulfides in the second, third, and fourth repeat and reduced cysteines in the first repeat. Disulfide formation in the first repeat limits the overall rate of oxidative refolding and contributes about half of the thermodynamic stability to native HcpB, estimated as 27 kJ mol(-1) at 25 degrees C and pH 7. The high contribution to stability of the first repeat may be explained by the repeat acting as a cap to protect the hydrophobic interior of the molecule.     

Exploring the folding pathway of green fluorescent protein through disulfide engineering

We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C‐termini is formed, folding is 2× faster, unfolding is 2000× slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m‐value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink‐induced perturbations in kinetics are useful for mapping the protein folding pathway.     

P22 tailspike trimer assembly is governed by interchain redox associations

Though disulfide bonds are absent from P22 tailspike protein in its native state, a disulfide-bonded trimeric intermediate has been identified in the tailspike folding and assembly pathway in vitro. The formation of disulfide bonds is critical to efficient assembly of native trimers as mutations at C-terminal cysteines reduce or inhibit trimer formation. We investigated the effect of different redox folding environments on tailspike formation to discover if simple changes in reducing potential would facilitate trimer formation. Expression of tailspike in trxB cell lines with more oxidizing cytoplasms led to lower trimer yields; however, observed assembly rates were unchanged. In vitro, the presence of any redox buffer decreased the overall yield compared to non-redox buffered controls; however, the greatest yields of the native trimer were obtained in reducing rather than oxidizing environments at pH 7. Slightly faster trimer formation rates were observed in the redox samples at pH 7, perhaps by accelerating the reduction of the disulfide-bonded protrimer to the native trimer. These rates and the effects of the redox system were found to depend greatly on the pH of the refolding reaction. Oxidized glutathione (GSSG) trapped a tailspike intermediate, likely as a mixed disulfide. This trapped intermediate was able to form native trimer upon addition of dithiothreitol (DTT), indicating that the trapped intermediate is on the assembly pathway, rather than the aggregation pathway. Thus, the presence of redox agents interfered with the ability of the tailspike monomers to associate, demonstrating that disulfide associations play an important role during the assembly of this cytoplasmic protein.     

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Effects of deletion and shift of the A20-B19 disulfide bond on the structure, activity, and refolding of proinsulin

To investigate the role of the A20-B19 disulfide bond in the structure, activity and folding of proinsulin, a human proinsulin (HPI) mutant [A20, B19Ala]-HPI was prepared. This mutant, together with another proinsulin mutant previously constructed with an A19Tyr deletion, which can also be taken as shifted mutant of the A20-B19 disulfide bond, were studied for their in vitro refolding, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion in comparison with native proinsulin. The results indicate that deletion of the A20-B19 disulfide bond results in a large decrease in the alpha-helix content of the molecule and higher sensitivity to tryptic digestion. Both the deletion and shift mutations, especially the latter, cause a great decrease in the biological activity of proinsulin analogues. The folding yields of HPI analogues were much lower than that of HPI. And the shift mutant, [Delta A19Tyr]-HPI, was scarcely refolded correctly in vitro and its refolding yield was extremely low. These results suggest that the A20-B19 disulfide bond plays an important role in the structural stabilization and folding of the insulin precursor. By summarizing the refolding studies on proinsulin, a possible folding pathway is proposed.     

pH-dependent stability of the human alpha-lactalbumin molten globule state: contrasting roles of the 6 - 120 disulfide and the beta-subdomain at low and neutral pH

Many proteins are capable of populating partially folded states known as molten globule states. Alpha-lactalbumin forms a molten globule under a range of conditions including low pH (the A-state) and at neutral pH in the absence of Ca(2+) with modest amounts of denaturant. The A-state is the most thoroughly characterized and thought to mimic a kinetic intermediate populated during refolding at neutral pH. We demonstrate that the properties and interactions that stabilize the A-state and the pH 7 molten globule of human alpha-lactalbumin differ. The unfolding of the wild-type protein is compared to the unfolding of a variant that lacks the 6 - 120 disulfide bond and to an autonomously folded peptide construct that we have previously shown represents the minimum core structure of the A-state of human alpha-lactalbumin. Studies conducted at pH 2 and 7 show that the disulfide makes little contribution to the stability of the molten globule at pH 7 but is important at pH 2. In contrast, the beta-subdomain of the protein is less important at pH 2 than at pH 7. The role of helix propensity in stabilizing the different forms of the molten globule state is examined and it is shown that it cannot account for the differences. The strikingly different behavior observed at pH 2 and 7 indicates that the A-state may not be a rigorous mimic of the folding intermediate populated at pH 7.     

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.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

The rough energy landscape of superfolder GFP is linked to the chromophore

Many green fluorescent protein (GFP) variants have been developed for use as fluorescent tags, and recently a superfolder GFP (sfGFP) has been developed as a robust folding reporter. This new variant shows increased stability and improved folding kinetics, as well as 100% recovery of native protein after denaturation. Here, we characterize sfGFP, and find that this variant exhibits hysteresis as unfolding and refolding equilibrium titration curves are non-coincident even after equilibration for more than eight half-lives as estimated from kinetic unfolding and refolding studies. This hysteresis is attributed to trapping in a native-like intermediate state. Mutational studies directed towards inhibiting chromophore formation indicate that the novel backbone cyclization is responsible for the hysteresis observed in equilibrium titrations of sfGFP. Slow equilibration and the presence of intermediates imply a rough landscape. However, de novo folding in the absence of the chromophore is dominated by a smoother energy landscape than that sampled during unfolding and refolding of the post-translationally modified polypeptide.     

Oxidative folding of human lysozyme: effects of the loss of two disulfide bonds and the introduction of a calcium-binding site

Mutant human lysozymes (HLZ) lacking two disulfide bonds were constructed to study the importance of each disulfide bond on oxidative refolding. To avoid destabilization, a calcium-binding site was introduced. Five of the six species of two-disulfide mutants could be obtained with enzymatic activity. Based on the information obtained from refolding and unfolding experiments, the order of importance in oxidative refolding was found to be as follows: SS2(Cys30-Cys116) > SS1(Cys6-Cys128) SS3(Cys65-Cys81) > SS4(Cys77-Cys95). Without SS2, these mutants refolded with low efficiency or did not refold at all. The bond SS2 is located in the interface of B-and D-helices, and a small hydrophobic cluster is formed near SS2. This cluster may play an important role in the folding process and stabilization, and SS2 may act as a stabilizer through its polypeptide linkage. The bond SS2 is the most important disulfide bond for oxidative folding of lysozymes.     

Tracking the unfolding and refolding pathways of outer membrane protein porin from Paracoccus denitrificans

We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

Folding of ribonuclease T1. 2. Kinetic models for the folding and unfolding reactions

The slow refolding of ribonuclease T1 was investigated by different probes. Structural intermediates with secondary structure are formed early during refolding, as indicated by the rapid regain of a native-like circular dichroism spectrum in the amide region. This extensive structure formation is much faster than the slow steps of refolding, which are limited in rate by the reisomerization of incorrect proline isomers. The transient folding intermediates were also detected by unfolding assays, which make use of the reduced stability of folding intermediates relative to that of the native protein. The results of this and the preceding paper [Kiefhaber et al. (1990) Biochemistry (preceding paper in this issue)] were used to propose kinetic models for the unfolding and refolding of ribonuclease T1. The unfolding mechanism is based on the assumption that, after the structural unfolding step, the slow isomerizations of two X-Pro peptide bonds occur independently of each other in the denatured protein. At equilibrium a small amount of fast-folding species coexists with three slow-folding species: two with one incorrect proline isomer each and another, dominant species with both these prolines in the incorrect isomeric state. In the mechanism for refolding we assume that all slow-folding molecules can rapidly regain most of the secondary and part of the tertiary structure early in folding. Reisomerizations of incorrect proline peptide bonds constitute the slow, rate-limiting steps of refolding. A peculiar feature of the kinetic model for refolding is that the major unfolded species with two incorrect proline isomers can enter two alternative folding pathways, depending on which of the two reisomerizes first. The relative rates of reisomerization of the respective proline peptide bonds at the stage of the rapidly formed intermediate determine the choice of pathway. It is changed in the presence of prolyl isomerase, because this enzyme catalyzes these two isomerizations with different efficiency and consequently leads to a shift from the very slow to the intermediate refolding pathway.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

Pressure-induced unfolding/refolding of ribonuclease A: static and kinetic Fourier transform infrared spectroscopy study

In this paper, we illustrate the use of high-pressure Fourier transform infrared (FT-IR) spectroscopy to study the reversible presssure-induced unfolding and refolding of ribonuclease A (RNase A) and compare it with the results obtained for the temperature-induced transition. FT-IR spectroscopy monitors changes in the secondary structural properties (amide I' band) or tertiary contacts (tyrosine band) of the protein upon pressurization or depressurization. Analysis of the amide I' spectral components reveals that the pressure-induced denaturation process sets in at 5. 5 kbar at 20 degrees C and pH 2.5. It is accompanied by an increase in disordered structures while the content of beta-sheets and alpha-helices drastically decreases. The denatured state above 7 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as an extended random coil. Increase of pH from 2.5 to 5.5 has no influence on the structure of the pressure-denatured state; it slightly changes the stability of the protein only. All experimental evidence indicates that the pressure-denatured states of monomeric proteins have more secondary structure than the temperature-denatured states. Different modes of denaturation, including pressure, may correlate differently with the roughness of the energy scale and slope of the folding funnel. For these reasons we have also carried out pressure-jump kinetic studies of the secondary structural evolution in the unfolding/refolding reaction of RNase A. In agreement with the theoretical model presented by Hummer et al. [(1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1552-1555], the experimental data show that pressure slows down folding and unfolding kinetics (here 1-2 orders of magnitude), corresponding to an increasingly rough landscape. The kinetics remains non-two-state under pressure. Assuming a two-step folding scenario, the calculated relaxation times for unfolding of RNase A at 20 degrees C and pH 2.5 can be estimated to be tau(1) approximately 0.7 min and tau(2) approximately 17 min. The refolding process is considerably faster (tau(1) approximately 0.3 min, tau(2) approximately 4 min). Our data show that the pressure stability and pressure-induced unfolding/refolding kinetics of monomeric proteins, such as wild-type staphylococcal nuclease (WT SNase) and RNase A, may be significantly different. The differences are largely due to the four disulfide bonds in RNase A, which stabilize adjacent structures. They probably lead to the much higher denaturation pressure compared to SNase, and this might also explain why the volume change of WT SNase upon unfolding is about twice as large.     

Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.