Refolding Of Ribonuclease Research Articles

Expanding Anfinsen’s Principle: Controlling Protein Structure by Altering Local Translation Rate

Anfinsen's seminal experiments on the refolding of ribonuclease in vitro showed that the final native structure of a protein is determined by its primary amino acid sequence. In vivo, a nascent polypeptide chain can start to fold co-translationally, and the conformations adopted during chain synthesis can be distinct from the conformations adopted by free, full length chains refolded after dilution from denaturant. Moreover, altering protein translation rate has been shown to further affect co-translational protein folding mechanisms. Translation rate can be altered without changing the encoded amino acid sequence by altering synonymous codon usage. But common codons are typically translated faster than rare codons, and clusters of rare codons can cause significant pauses in translation. Here, we designed and tested a system to investigate the effects of a cluster of synonymous rare codons on a protein folding mechanism and its final folded structure. We designed a protein that consists of three half-domains, connected by flexible linkers. The N- and C-terminal half-domains compete to interact with the central half-domain. This protein therefore has the potential to form one of two mutually exclusive native structures. We altered synonymous codons at the 5’ end of the sequence encoding the C-terminal half-domain to adjust the rate at which the C-terminal half-domain appears outside the ribosome exit tunnel. The presence of rare codons at this location causes the N-terminal half-domain to pair with the central domain more often than the C-terminal half-domain. This effect is tunable, based on the rareness of the codons and the corresponding duration of the translation pause. These results suggest that Anfinsen's principle might need to be expanded, to include possible effects of translation rate on protein structure formation. Either

The Folding Pathway of Onconase Is Directed by a Conserved Intermediate

A promising approach to unravel the relationship between sequence information, tertiary structure, and folding mechanism of proteins is the analysis of the folding behavior of proteins with low sequence identity but comparable tertiary structures. Ribonuclease A (RNase A) and its homologues, forming the RNase A superfamily, provide an excellent model system for respective studies. RNase A has been used extensively as a model protein for folding studies. However, little is known about the folding of homologous RNases. Here, we analyze the folding pathway of onconase, a homologous protein from the Northern leopard frog with great potential as a tumor therapeutic, by high-resolution techniques. Although onconase and RNase A significantly differ in the primary structure (28% sequence identity) and in thermodynamic stability (DeltaDeltaG = 20 kJ mol(-1)), both enzymes possess very similar tertiary structures. The present folding studies on onconase by rapid mixing techniques in combination with fluorescence and NMR spectroscopy allow the structural assignment of the three kinetic phases observed in stopped-flow fluorescence spectroscopy. After a slow peptidyl-prolyl cis-to-trans isomerization reaction in the unfolded state, ONC folds via an on-pathway intermediate to the native state. By quenched-flow hydrogen/deuterium exchange experiments coupled with 2D NMR spectroscopy, 31 amino acid residues were identified to be involved in the structure formation of the intermediate. Twelve of these residues are identical in the RNase A sequence, which is a significantly higher percentage (39%) than the overall 28% sequence identity. Moreover, the structure of this intermediate closely resembles two of the intermediates that occur early during the refolding of RNase A. Obviously, in spite of considerable differences in their amino acid sequence the initial folding events of both proteins are comparable, guided by a limited number of conserved residues.

DIVINING PROTEIN ARCHITECTURE

EVER SINCE THE 1960S, WHEN experiments on the refolding of ribonuclease by the late biochemist Christian B. Anfinsen demonstrated that a protein's amino acid sequence determines its three-dimensional structure, researchers have been working toward using sequence information as a basis for predicting protein structure. Applications of protein structure prediction include learning more about protein folding, designing functional proteins from scratch, and divining the structure and function of proteins from living organisms. Five or six years ago, the protein structure prediction problem was considered to be pretty much unsolved. But since then considerable progress has been made: Scientists can now construct fairly accurate models that show where a protein's chain is supposed to go, and many of the topological features of these models closely resemble those in actual structures. Not that all the problems have now been solved. In terms of actually explaining protein folding from first principles, I think w...

Hydrogen Peroxide-induced Structural Alterations of RNase A

Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H(2)O(2) exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time- and H(2)O(2) concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H(2)O(2) concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.

Turn scanning. Experimental and theoretical approaches to the role of turns.

AbstractThe mechanism that enables a protein to fold to its correct three-dimensional conformation—given only the information available from its sequence-has been a matter of intense investigation since Anfinsen’s classical experiment on the refolding of ribonuclease almost 40 years ago (1). The importance of turn sequences in the kinetic and thermodynamic aspects of protein folding remains a controversial point, although their role—especially forβ-turns—has been regarded as significant (i.e., 2–10). What role, if any, do turn sequences play in determining the native structures of proteins? For the experimentalist, this question is usually rephrased as: to what extent can substitution, insertion, and deletion be tolerated at turn positions before kinetic and/or thermodynamic properties are affected? It is indeed surprising how few experimental studies have been performed to carefully investigate the role of turns. To the theoretician, this question of the importance of turns revolves around the ability of computational studies to attempt to determine the propensity of isolated small peptides, as well as larger structures, to form turns. While much research has been conducted in this field, even here the answers remain uncertain. KeywordsFolding ProcessDenaturant ConcentrationUmbrella SamplingTurn RegionTurn StructureThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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.

Reactivity of glutaredoxins 1, 2 and 3 from Escherichia coli and protein disulfide isomerase towards glutathionyl-mixed disulfides in ribonuclease A

We have examined the activity of protein disulfide isomerase (PDI) and glutaredoxin (Grx) 1, 2 and 3 from Escherichia coli to catalyze the cleavage of glutathionylated ribonuclease A (RNase-SG) by 1 mM GSH to yield reduced RNase. Apparent K m values for RNase-SG were similar, 2–10 μM, for Grx 1, 3 and PDI but Grx 1 and Grx 3 showed 500-fold higher turnover numbers than PDI. The atypical Grx 2 also catalyzed deglutathionylation by GSH, but had higher K m and apparent turnover number values compared to the two classical Grx. Refolding of RNase in a glutathione redox buffer was catalyzed by PDI. However, it could be measured only after a characteristic lag phase that was shortened by all three E. coli Grxs in a concentration-dependent manner. A role of the glutaredoxin mechanism in the endoplasmic reticulum is suggested.

Effect of glutaredoxin and protein disulfide isomerase on the glutathione-dependent folding of ribonuclease A.

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase A (rd-RNase A) and mixed disulfide between glutathione and reduced RNase A derivative (GS-RNase A) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ESMS) time-course analysis and recovery of the native catalytic activity. The ESMS analysis permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding of rd-RNase A and GS-RNase A was also performed in the presence of glutaredoxin (Grx) and/or protein disulfide isomerase (PDI). All the analyses indicate a pathway of sequential reactions in the formation of native RNase A. First, the reduced protein reacts with a single glutathione molecule to form a mixed disulfide which then evolves to an intramolecular S-S bond via thiol-disulfide exchange. Only at this stage, the intermediate containing one intramolecular S-S reacts with a further glutathione molecule, reiterating the process. An analogous mechanism occurs in the refolding of GS-RNase A. The structural analysis of the intermediates formed during the refolding of RNase A showed for the first time that Grx is actually able to catalyze both formation and reduction of mixed disulfides involving glutatione. In both refolding processes, starting from either rd-RNase A or GS-RNase A, Grx displays a significant catalysis at the early stages of the process. Addition of PDI led to a net catalysis of the entire process without appearing to alter the refolding pathway. In the presence of both Grx and PDI, the two enzymes showed a synergistic activity either starting from rd-RNase A, as previously reported [Lundström, J., and Holmgren, A. (1995) J. Biol. Chem. 270, 7822-7828], or starting from GS-RNase A. Present data suggest that the synergistic effect can be explained assuming that Grx actually facilitates PDI action by catalyzing formation or reduction of mixed disulfides. The mixed disulfides are then rapidly converted into intramolecular disulfides in the presence of PDI. These steps are repeated sequentially throughout the whole refolding, resulting in an immediate formation of fully oxidized species even at the very beginning of the reaction. Finally, a Grx mutant, C14S Grx, in which one of the active site cysteine residues (Cys14) had been replaced by serine, had a similar effect on the distribution of folding intermediates, compared to the wild-type protein, thus demonstrating that Grx acts by a monothiol mechanism either in the reduction or in the oxidation step.

Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

Kinetic dynamic light scattering is a useful tool to follow compaction during protein folding. In contrast to measurements of the formation of secondary structure and side chain ordering, kinetic measurements of compactness are not well established up to now. This work describes the adaptation of a stopped-flow system (SFM-3) to a dynamic light scattering apparatus, which allows one to monitor the compaction of protein molecules by measuring the hydrodynamic Stokes radius R. The feasibility of such investigations was demonstrated by measuring R and the integrated scattered intensity I during refolding of ribonuclease A and phosphoglycerate kinase from yeast. Refolding was initiated by rapid mixing of protein solutions containing high concentrations of guanidine hydrochloride with buffer. Between 20 and 50 mixing events were performed in these experiments. Measuring both R and I in one and the same experiment is important to distinguish between true folding of individual molecules and cases where folding is accompanied by the appearance of transient oligomers or associated misfolded structures. On refolding of ribonuclease A (0.6 M GuHCl, 25 °C), after a fast phase the Stokes radius decreased from 2.26 nm to 1.95 nm with a time constant of 27 s without detectable aggregates. By contrast, transient and stable oligomers have been observed during the more complex folding of phosphoglycerate kinase. In general, the time-resolution of the method is of the order of 1 s. It can be extended to the subsecond time-range if the number of shots is not limited by the amount of protein available.

Refolding of thermally and urea-denatured ribonuclease A monitored by time-resolved FTIR spectroscopy.

We undertook a first detailed comparative analysis of the refolding kinetics of ribonuclease A (RNase A) by time-resolved Fourier transform infrared spectroscopy. The refolding process was initiated either by applying a temperature jump on the thermally denatured protein or by rapid dilution of a concentrated [13C]urea solution containing the chemically unfolded protein. The dead time of the injecting and mixing devices and the time-resolution of the spectrometer permitted us to monitor the refolding kinetics in a time range of 100 ms to minutes. The infrared amide I' band at 1631 cm-1 was used to directly probe the formation of beta-sheet structure during the refolding process. The aromatic ring stretching vibration of tyrosine at 1515 cm-1 was employed as a local monitor that detects changes in the tertiary structure along the folding pathway. The comparative analysis of the kinetics of the beta-sheet formation of chemically and thermally denatured ribonuclease A revealed similar folding rates and amplitudes when followed under identical refolding conditions. Therefore, our kinetic infrared studies provide evidence for a high structural similarity of urea-denatured and heat-denatured RNase A, corroborating the conclusions derived from the direct comparison of the infrared spectra of thermally and chemically denatured RNase A under equilibrium conditions [Fabian, H., & Mantsch, H.H. (1995) Biochemistry 34, 13651-13655]. In detail, the kinetic infrared data demonstrate that in the time window of 0.1-30 s approximately 40% of the native beta-sheet structure in RNase A is formed in the presence of 0.6 M urea at pH* 3.6, indicating that up to 60% of the beta-structure is formed out of the time window used in this study. Temperature jump experiments in the absence of chemical denaturants exhibited faster and more complex refolding kinetics. In addition, differences in the time constants of refolding derived from the amide I' band at 1631 cm-1 and from the tyrosine vibration at 1515 cm-1 were observed, indicating that the formation of secondary structure precedes the formation of stable tertiary contacts during the refolding of RNase A.

Temperature-jump-induced refolding of ribonuclease A: A time-resolved FTIR spectroscopic study

FTIR difference spectroscopy has been used for the first time to investigate the kinetics of secondary structure formation during refolding. The refolding process of ribonuclease A (RNase A) as a model system was induced by applying a temperature-jump of 60 degrees. The temperature-jump was triggered by rapidly injecting a small volume of the thermally unfolded protein solution at 80°C into a special cuvette system kept at 20°C. The dead-time of the injection and the time resolution of the FTIR spectrometer permitted the observation of refolding processes in a time window ranging from 170 ms to several minutes. Specifically, the formation of β-structures and the disappearance of irregular conformations could be observed in this time interval.

Refolding of RNase A at high concentrations: identification of non-native species

In this paper, we present an analysis of the soluble species formed on refolding of RNase A at various concentrations, in order to characterize these species with respect to structure and activities. Studies were carried out using reverse-phase high-performance liquid chromatography, circular dichroism, chromatography and ultracentrifugation. At all concentrations of protein used, RNase A refolded to the native form, together with formation of non-native species. These non-native species are either misfolded monomers or aggregates; the percentage of such species increases with increasing concentration of enzyme. Such aggregation appears to be a non-random process governed by intermolecular disulfide crosslinking between monomers. These results reaffirm the principle that the information for folding of the protein is encoded in the amino acid sequence itself.

Kinetic Models for Unfolding and Refolding of Ribonuclease T 1 with Substitution of Cis-proline 39 by Alanine

The replacement of cis proline 39 of ribonuclease T 1 by an alanine residue leads to a decrease in stability by about 20 kJ/mol and to major changes in the folding kinetics that are not easily explained by the proline model for protein folding. In particular, a novel very slow reaction is observed in the refolding of the Pro39Ala variant. Here the unfolding and refolding kinetics of this protein are further investigated. We show that the very slow reaction is not a prolyl isomerization. It is not created by a slow isomerization of the unfolded protein, nor is it catalyzed by prolyl isomerase, and all molecules have to undergo this reaction during refolding. Most of the unfolded Pro39Ala molecules contain an incorrect trans isomer at the remaining cis Pro55. They use a sequential pathway for refolding, in which trans to cis isomerization at Pro55 precedes the very slow reaction. The refolding of the minor fraction of unfolded Pro39Ala molecules with a correct cis isomer at proline 55 is a single first-order reaction that is limited in rate by the very slow step. The folding mechanism of wild-type ribonuclease T 1 cannot be used to explain these results and independent mechanisms are proposed to model the unfolding and refolding of the Pro39Ala variant. The molecular interpretation of the changes in the folding mechanism is tied to the question, as to whether the cis character of the peptide bond at position 38-39 is maintained after the substitution of Pro39 by alanine. A possible explanation could be that the novel very slow folding reaction involves the trans to cis isomerization of the Tyr38-Ala39 bond. Such a reaction is probably slow, since the activation energy is high and since tight coupling with the formation of structure is required to stabilize the cis form of a non-prolyl peptide bond. Alternatively, the strong decrease in folding rate could be correlated with the general destabilization of ribonuclease T 1 by the Pro39Ala mutation.

Stability and Folding Kinetics of Ribonuclease T 1 are Strongly Altered by the Replacement of Cis-proline 39 with Alanine

The refolding of ribonuclease T 1 involves two major slow processes that exhibit properties of prolyl isomerization reactions. A comparison of the wild-type protein and a designed variant where the cis Ser54-Pro55 bond was replaced by a Gly54-Asn55 bond indicated that the faster of these reactions is the isomerization of Pro55. Here we report the replacement of the other cis proline of ribonuclease T 1 at position 39 by alanine. The Pro39Ala variant is similar to the wild-type protein in secondary and tertiary structure, and the enzymatic activity towards RNA and a dinucleotide substrate remains almost unchanged. The fluorescence emission of the single Trp59 is lowered by the Pro39Ala substitution, probably because Trp59 is in close contact to Pro39 in wild-type ribonuclease T 1. Unlike the substitution of cis Pro55, the Pro39Ala mutation is strongly destabilizing and reduces the Gibbs free energy of the folded protein by about 20 kJ/mol. Pro39 is buried in native RNase T 1 and located near the active site. The observed destabilization could originate from the presence of a cis alanyl bond in the Pro39Ala variant or from a local distortion caused by the incorporation of a trans alanyl peptide bond in the interior of the protein. In the refolding kinetics the replacement of Pro39 leads to a disappearance of the fast-refolding species. Refolding still involves two consecutive slow steps. The first and faster step could be the isomerization of the remaining cis Pro55. The second, very slow step is a novel reaction that appears to have no counterpart in the refolding of the wild-type protein. All mutant molecules must undergo this reaction before reaching the native state. These major changes in the folding kinetics strongly indicate that cis-Pro39 is indeed of major importance for the folding of the wild-type protein. They indicate, moreover, that some new feature of protein folding kinetics is observed in these studies of the Pro39Ala variant.

A carboxypeptidase Y pulse method to study the accessibility of the C-terminal end during the refolding of ribonuclease A.

Carboxypeptidase Y pulses, applied after various times of refolding, were employed to probe the accessibility of the C-terminus of RNAase A during the refolding process. The increase in resistance against proteolytic cleavage was measured by determination of the amount of liberated C-terminal amino acids and by activity assays. The results indicate that the C-terminus of RNAase becomes inaccessible early in the course of refolding, if folding is carried out at low temperatures under conditions that effectively stabilize the native state. At higher temperatures (25 degrees C) or under conditions of marginal stability, intermediates are not populated and protection against proteolytic cleavage is not detectable before the formation of the native state. The method described may be used to monitor the accessibility of the C-terminus of various proteins during refolding. However, intermediates on the folding pathway can only be observed if the native state is stable against carboxypeptidase attack.

Separation of the nativelike intermediate from unfolded forms during refolding of ribonuclease A.

In an effort to determine structural properties of the nativelike intermediate (i.e., IN) which forms during the refolding of RNase A, refolding samples were subjected to rapid HPLC gel filtration which allowed us to separate IN from unfolded forms of RNase. The comparison of these samples, enriched in IN and depleted of unfolded forms, with unseparated control samples at the same stage of refolding allowed certain conclusions to be drawn concerning the properties of IN. First, the results show that the transition from IN to native RNase occurs with only small changes in fluorescence. This means that the major fluorescence changes seen during normal refolding experiments must be associated with changes in proline isomerization of unfolded species and/or with the refolding step itself but not with the IN----N step. Second, the fluorescence assay for isomerization of proline-93 shows that IN exists with proline-93 in a state of isomerization identical with or very similar to native RNase; i.e., proline-93 is cis in IN and not trans as suggested by others. All results are semiquantitatively consistent with our earlier refolding model and not nearly so consistent with alternative models which assume that most or all of the slow-refolding forms of RNase have proline-93 in the incorrect trans state.

Intermediates in the refolding of ribonuclease at subzero temperatures. 1. Monitoring by nitrotyrosine absorbance.

Derivatives of ribonuclease A in which tyrosines-73, -76, and -115 were nitrated have been synthesized, purified to homogeneity, and characterized by NMR, isoelectric points, absorbance spectra, and catalytic activity. The positions of their reversible thermal unfolding transitions were determined in 35% methanol at pH* 3.0 and 6.0. In the present study the kinetics of the refolding of these nitrotyrosine derivatives were measured at -15 degrees C at pH* 3.0 and 6.0 by using a cryosolvent composed of 35% aqueous methanol. The rates of folding of different regions of the molecule were determined by using the nitrotyrosines as environmentally sensitive probes. Multiphasic kinetics were observed for the refolding of the nitro-Tyr-115, -73, and -76 derivatives. The native environment about Tyr-115 was formed more rapidly than that about Tyr-73 and -76, and the native environment about both these tyrosines was attained much sooner than the native state itself, as judged by other probes. The results indicate that different regions of the molecule attain their native environments at different rates. This observation shows that the folding pathway must involve partially folded intermediate states.

Evidence for the existence of three or more slow phases in the refolding of ribonuclease A and some characteristics of the phases.

The slow refolding kinetics of RNase A have been analyzed, by using a nonlinear least-squares program for deconvoluting the kinetic phases and applying statistical tests for quality of fit. It is found that a minimum of three slow phases are required to fit the kinetic data properly, and this is true whether the method of detection is absorbance of fluorescence. Since the number of phases and the relaxation times for each phase are independent of the method of detection, it is concluded that the same three rate-limiting processes are seen by absorbance and fluorescence. These phases correspond to the XY, CT, and ct phases described in our earlier studies. The fact that fluorescence-detected kinetics are somewhat slower than absorbance-detected kinetics is a trivial effect due not to differences in relaxation times but to the fact that the amplitude of the CT phase is enhanced in fluorescence measurements, at the expense of the faster XY phase, because of intrinsic fluorescence changes associated with the isomerization of proline-93. By use of a new double-jump technique [Schmid, F.X., Grafl, R., Wrba, A., & Beintema, J.J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 872], it is shown that proline-93 isomerizes as the rate-limiting step in only one of the three phases, the CT phase, and that this phase involves only 25-30% of the RNase molecules. There is still no indication as to the molecular events that occur in the large, ammonium sulfate dependent XY phase, which is the pathway for formation of the nativelike intermediate.

Refolding of ribonuclease in the presence and absence of ammonium sulfate pulses. Comparison between experiments and simulations.

Experiments have been carried out on ribonuclease A in which refolding in high concentrations of guanidine hydrochloride is either preceded or not preceded by a short ammonium sulfate pulse. Application of the pulse causes the rapid formation of the nativelike intermediate, and the effect of this pulse was determined by using three different methods for monitoring the subsequent refolding reaction: direct absorbance, direct fluorescence, and a double-jump fluorescence unfolding assay which is specific for the isomerization of proline-93. The effect of the pulse is quite different depending on the method of detection. With absorbance detection, the pulse causes a large reduction in the refolding amplitude with no change in the kinetics of the decay curve, while with the fluorescence unfolding assay, the pulse causes no change in the refolding amplitude but produces a large acceleration in the decay kinetics. The results with direct fluorescence are intermediate with some reduction seen in the refolding amplitude and some acceleration in the decay kinetics. The results of these experiments are simulated by using the simple model of Lin and Brandts (1984) [Lin, L.-N., & Brandts, J. F. (1984) Biochemistry 23, 5713] in which proline-93 must be in the correct cis configuration before folding to the native or nativelike state can occur. In all cases, the simulations accurately predict the experimental results for all three methods of detection, without any adjustment of parameter values from those published earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

Proline isomerization during refolding of ribonuclease A is accelerated by the presence of folding intermediates

The trans → cis isomerization of Pro 93 was measured during refolding of bovine ribonuclease A. This isomerization is slow (τ = 500s) under marginally stable folding conditions of 2.0 M GdmCl, pH 6, at 10°C. However, it is strongly accelerated (τ = 100 s) in samples which, prior to isomerization, had been converted to a folding intermediate by a 15s refolding pulse under strongly native conditions (0.8 M ammonium sulfate, 0°C). The results demonstrate that extensive folding is possible before Pro 93 isomerizes to its native cis state and that the presence of structural folding intermediates leads to a marked increase in the rate of subsequent proline isomerization.