Location of the antigenic determinants of bovine pancreatic ribonuclease.

Four antigenic regions of native bovine pancreatic ribonuclease have been located by using antibodies that react specifically with segments 1--13, 31--79, and 80--124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicates that there are at least three antigenic determinants to which antibody molecules can bind simultaneously in the presence of excess antibodies. Analysis of binding data, however, for each purified specific antibody preparation, carried out by the method of Berzofsky et al. [Berzofsky, J. A., Curd, J. G., & Schechter, A. N. (1976) Biochemistry, 15, 2113], leads to an estimate of four for the number of antigenic determinants in ribonuclease; this estimate had also been made earlier by Stelos et al. [Stelos, P., Fothergill, J. E., & Singer, S. J. (1960) J. Am. Chem. Soc. 82, 6034]. We find that one determinant is associated with each of segments 1--13 and 80--124 and two with segment 31--79. No antigenic activity could be detected for segment 14--29 either in native ribonuclease or in the free fragment. These conclusions are based on (1) the use of specific peptides to isolate purified antibodies by affinity chromatography, (2) immunoprecipitation of an antigenic peptide from the peptic digest of ribonuclease, (3) competitive inhibition studies with various peptide and protein fragments [cyanogen bromide fragments 1--13, 31--79, and 80--124, the tryptic peptides 40--61 and 105--224, S-peptide, S-protein, and des(121--124)-RNase], and (4) comparison and evaluation of the published effects on antigenicity of chemical and enzymatic modifications and changes in sequence among homologous ribonucleases. These approaches provide evidence that the four antigenic determinants are localized around the alpha-helical portion of segment 1--10, somewhere in segment 40--61, at the beta bend in segment 63--75, and either at the beta bend or beta sheet in segment 87--104 of native ribonuclease.     

Impact of an easily reducible disulfide bond on the oxidative folding rate of multi-disulfide-containing proteins.

The burial of native disulfide bonds, formed within stable structure in the regeneration of multi-disulfide-containing proteins from their fully reduced states, is a key step in the folding process, as the burial greatly accelerates the oxidative folding rate of the protein by sequestering the native disulfide bonds from thiol-disulfide exchange reactions. Nevertheless, several proteins retain solvent-exposed disulfide bonds in their native structures. Here, we have examined the impact of an easily reducible native disulfide bond on the oxidative folding rate of a protein. Our studies reveal that the susceptibility of the (40-95) disulfide bond of Y92G bovine pancreatic ribonuclease A (RNase A) to reduction results in a reduced rate of oxidative regeneration, compared with wild-type RNase A. In the native state of RNase A, Tyr 92 lies atop its (40-95) disulfide bond, effectively shielding this bond from the reducing agent, thereby promoting protein oxidative regeneration. Our work sheds light on the unique contribution of a local structural element in promoting the oxidative folding of a multi-disulfide-containing protein.     

Intermediates in the refolding of reduced ribonuclease A.

The intermediates with one, two, three or four disulphide bonds which accumulate during unfolding of native ribonuclease and refolding of the reduced protein have been trapped by rapid alkylation with iodoacetate and separated by ionexchange chromatography. They have been characterized to varying extents by their enzymic activity, electrophoretic mobility through polyacrylamide gels, disulphide bonds between cysteine residues, the environments of the six tyrosine residues as indicated by ultraviolet absorption and fluorescence spectra, interaction with antibodies directed against either the trapped unfolded reduced protein or the native folded protein, and for the disruption by urea of any stable conformation producing a change in molecular shape.Correctly refolded ribonuclease was indistinguishable from the original native protein, but virtually all the intermediates with up to four disulphide bonds formed directly from the reduced protein were enzymically inactive and unfolded by these criteria. Unfolding of native ribonuclease was an all-or-none transition to the fully reduced protein, with no accumulation of disulphide intermediates. The intermediates in refolding are separated from the fully folded state by the highest energy barrier in the folding transition; they may be considered rapidly interconvertible, relatively unstable microstates of the unfolded protein. The measured elements of the final conformation are not acquired during formation of the first three disulphide bonds, but appear simultaneously with formation of the fourth native disulphide bond.These observations with ribonuclease are qualitatively similar to those made previously in greater detail with pancreatic trypsin inhibitor and suggest a possible general pattern for the kinetic process of protein unfolding and refolding.     

Solution structure of the isolated ribonuclease C-terminal 112-124 fragment

The conformational properties of the ribonuclease C-terminal 112-124 fragment have been studied by CD and 1H- and 13C-NMR in an attempt to determine whether native secondary structure elements other than alpha-helices have stability enough to be detected when isolated in aqueous solution. Only sequential alpha N and intraresidue NOE cross-peaks are observed in the NOESY spectra, a fact which points towards an essentially extended polypeptidic chain. Observed spectral variations with temperature, pH and urea addition allowed the identification of two non-random regions within the chain. The first one is located within residues 119-121, the same region where a native salt bridge (H119...D121) exists in the native protein, and the stability of that structure is affected by the protonation state of carboxylate groups. The second one involves the S123 and V124 residues at the C-terminal end. No signs of the native 112-115 beta-turn were detected which suggests that, in contrast to alpha-helices, long range interactions may be needed to stabilize these secondary structure elements.     

Low energy pathways and non-native interactions: the influence of artificial disulfide bridges on the mechanism of folding

Four versions of a beta-sheet protein (CD2.d1) have been made, each with a single artificial disulfide bond inserted into hairpin structures. Folding kinetics of reduced and oxidized forms shows bridge position strongly influences its effect on the folding reaction. Bridging residues 58 and 62 does not affect the rapidly formed intermediate (I) or rate-limiting transition (t) state, whereas bridging 33 and 38, or 31 and 41, lowers the t-state energy, with the latter having the stronger influence. Bridging residues 79 and 90 stabilizes both I- and t-states. To assess additivity in the energetic effects of these bridges, four double-bridge variants have also been made. All show precise additivity of overall stability, with two showing additivity when ground states and the rate-limiting t-state are assessed, i.e. no measurable change in the folding mechanism occurs. However, combining 31-41 and 79-90 bridges produces a molecule that folds through a different pathway, with a much more stable intermediate than expected and a much higher t-state barrier. This is explained by the artificial introduction of stabilizing, non-native contacts in the I-state. More surprisingly, for another double-bridge version (58-62 and 79-90) both I- and t-states are less stable than expected, showing that conformational constraints introduced by the two bridges prevent formation of non-native contacts that would otherwise stabilize the I- and t-states, thereby lowering the energy of the folding landscape in the wild-type (unbridged) molecule. We conclude that the lowest energy path for folding has I- and t-state structures that are stabilized by non-native interactions.     

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.     

Study of protein topography with flash photolytically generated nonspecific surface-labeling reagents: surface labeling of ribonuclease A

A method for nonspecifically labeling essentially all exposed residues of a protein is described. A reactive aryl nitrene is generated from N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-Taurine), within 500 mus by flash photolysis in the presence of protein. The reactive nitrene is inserted in about 2 ms into those carbon-hydrogen bonds of the protein that are exposed to the solvent. The method is applied here to ribonuclease A to demonstrate the different degree of labeling of the native and denatured protein. On the basis of amino acid analysis, it appears that residues of the native protein that are buried in the interior of the molecule (as judged from the x-ray structure) do not react with the nitrene. However, when these residues (even nonreactive ones such as valine and proline) are exposed by denaturation of the protein, they do react with the nitrene. It is shown that native ribonuclease A retains 90% of its enzymatic activity when flashed in the absence of NAP-Taurine. This small loss in activity arises from the disruption of a limited portion of the native enzyme structure, as judged by circular dichroism, ultraviolet, and Raman spectra. The site of this limited disruption may be a portion of the enzyme surface near the Cys-26-Cys-84 disulfide bond. The utility of this surface labeling technique for studying the pathways of protein folding or unfolding is discussed.     

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol.

We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42 degrees C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Th1) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Th1 maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-delta Tm 16 degrees C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Th1 is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Th1 retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 31-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a beta-strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Th1 and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.     

A new two-disulphide intermediate in the refolding of reduced bovine pancreatic trypsin inhibitor

The apparently complete refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) is shown to produce a mixture of two species. One of these is native BPTI, but the other lacks the disulphide bond between cysteines 30 and 51. The latter species has a folded conformation very like that of native BPTI, and is oxidized by air to native BPTI on warming in aqueous solution. The two unreactive cysteine thiol groups appear to be buried in the interior of the molecule, which restricts access by reagents that can alkylate them or oxidize them to form the disulphide bond. The implications of this intermediate and its conformation for the understanding of protein folding are discussed.     

Folding kinetics of mammalian ribonucleases

The folding kinetics of seven different pancreatic ribonucleases are compared both under native conditions and within the unfolding transition. In general, the folding kinetics of these proteins are similar despite numerous amino acid substitutions. Ribonucleases with 4-6 proline residues show 80% slow-folding species. For three ribonucleases with 7 prolines this number increases to 90%. Porcine ribonuclease with a unique Pro 114-Pro 115 sequence folds significantly slower than other ribonucleases which do not show this sequence.     

Early steps in the refolding reaction of reduced ribonuclease A.

The early part of the reaction of refolding of reduced ribonuclease A has been studied using the reappearance of enzymatic activity as an index of refolding. It is found that a low level of activity, about 0.04% of that of native enzyme, can be measured early after refolding has been initiated. This low level of activity is apparently not due to a contaminant or to incompletely reduced RNAase A molecules, but rather seems to be a property of the bulk of the reduced protein. Furthermore, this early activity is sensitive to the reaction with N-ethyl-maleimide, showing that it is due to completely or partially reduced molecules. The amount of protein responsible for this early activity represents a small fraction of the total reduced RNAase A, and possesses binding properties similar to those of the native enzyme towards a substrate, 2′, 3′ CMP and an inhibitor, 2′ CMP. These results are interpreted as evidence for the existence of an equilibrium between native and unfolded conformations in reduced RNAase A, and are discussed with respect to the protein folding mechanism.     

Disulfide bonds and protein folding

The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of "rugged funnel" models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulfide bonds suggests an analogous scenario for conformational folding. Finally, engineered covalent cross-links may be used to assay for the association of protein segments in the folding transition state, as illustrated with RNase A.     

Significance of the four carboxyl terminal amino acid residues of bovine pancreatic ribonuclease A for structural folding

The C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) form a core structure in the initial stage of the folding process that leads to the formation of the tertiary structure. In this paper, roles of the C-terminal four amino acids in the structure, function, and refolding were studied by use of recombinant mutant enzymes in which these residues were deleted or replaced. Purified mutant enzymes were analyzed for their secondary structure, thermal stability, and ability to regenerate from the denatured and reduced state. The C-terminal deleted mutant enzymes showed lower hydrolytic activity for C>p and nearly identical CD spectra compared with the wild-type enzyme. The rate of recovery of activity was significantly different among the C-terminal deleted mutant enzymes when air oxidation was employed in the absence of GSH and GSSG: the rates decreased in the order of des-124-, des-(123-124)-, and des-(122-124)-RNase A. It is noteworthy that the regeneration rates of mutant RNase A in the presence of GSH and GSSG were nearly the same. Des-(121-124)-RNase A failed to recover activity both in the presence and absence of glutathione, due to the mismatched formation of disulfide bonds. The mutant enzyme in which all of the C-terminal four amino acid residues were replaced by alanine residues showed lower hydrolytic activity and an indistinguishable CD spectrum compared with the wild-type enzyme, and also recovered its activity from the denatured and reduced state by air oxidation. The D121 mutant enzymes showed decreased hydrolytic activity and identical CD spectra compared with the wild type. The recovery rates of activity of D121A and D121K were determined to be lower than that of the wild-type enzyme, while the rate of recovery of D121E was comparable to that of the wild type. The C-terminal amino acids play a significant role in the formation of the correct disulfide bonds during the refolding process, and the interaction of amino acid residues and the existence of the main chain around the C-terminal region are both important for achieving the efficient packing of the RNase A molecule.     

Non-redox-active small-molecules can accelerate oxidative protein folding by novel mechanisms

Multi-disulfide-bond-containing proteins acquire their native structures through an oxidative folding reaction which involves formation of native disulfide bonds through thiol-disulfide exchange reactions between cysteines and disulfides coupled to a conformational folding event. Oxidative folding rates of the four-disulfide-bond-containing protein bovine pancreatic ribonuclease A (RNase A) in the presence of the synthetic redox-active molecule, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), and in combination with non-redox-active trimethylamine-N-oxide (TMAO), and trifluorethanol were determined by HPLC analysis. The data indicate that regeneration of RNase A is enhanced 2-fold by BMC (50 microM) and 3-fold upon addition of TMAO (0.2 M) and TFE (3% v/v) relative to control experiments performed in the absence of small-molecules. Examination of the native tendency of the fully-reduced polypeptide and the stability of key folding intermediates suggests that the increased oxidative folding rate can be attributed to native-like elements induced within the fully-reduced polypeptide and the stabilization of native-like species by added non-redox-active molecules.     

Solution structure and dynamics of ribonuclease Sa.

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.     

Influence of an extrinsic cross-link on the folding pathway of ribonuclease A. Kinetics of folding-unfolding

The kinetics of folding/unfolding of cross-linked Lys7-dinitrophenylene-Lys41-ribonuclease A were studied and compared to those of unmodified ribonuclease A (RNase A) at various concentrations of guanidine hydrochloride. The folding of the denatured cross-linked protein involved one fast-folding species (22 +/- 4%) and two slow-folding species, as observed in unmodified ribonuclease A. Also, a nativelike intermediate, analogous to that reported previously for unmodified ribonuclease A [Cook, K. H., Schmid, F. X., & Baldwin, R. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6157], has been detected on the folding pathway of cross-linked ribonuclease A. The extrinsic cross-link between Lys7 and Lys41 did not affect the rate constants for the folding kinetics of these three species. The cross-link did, however, significantly affect the rate constant for unfolding of the native protein. The conformation of the protein in the transition state of the unfolding pathway was deduced from an analysis of the kinetic data. It appears that the 41 N-terminal residues are unfolded in the transition state of the unfolding pathway. Thus, the unfolding pathway of RNase A is sequential in that further unfolding (after the transition state) follows the unfolding of the 41 N-terminal residues. Also, the conformation of the 41 N-terminal residues does not play a role in the folding pathway. Presumably, if the cross-link were introduced instead between two other residues that are in the segment(s) involved in the rate-limiting step(s), it could increase the refolding rate constants and possibly the concentration of fast-folding species.     

Local interactions favor the native 8-residue disulfide loop in the oxidation of a fragment corresponding to the sequence Ser-50-Met-79 derived from bovine pancreatic ribonuclease A

A 30-residue peptide was obtained from ribonuclease A by chemical cleavage with cyanogen bromide, subsequent sulfitolysis with concomitant S-sulfonation, and finally enzymatic cleavage withStaphylococcus aureus protease. The peptide was converted to the free thiol form by reductive cleavage of the S-sulfo-protecting groups withd,l-dithiothreitol. This peptide consisted of residues 50–79 of the native sequence of ribonuclease A, with the exception that methionine-79 had been converted to homoserine. Included in this sequence are residues cysteine-65 and cysteine-72, which form a disulfide bond in the native enzyme, as well as cysteine-58. This molecule may form one of three possible intramolecular disulfide bonds upon thiol oxidation, viz. one loop of 15 and 2 of 8 residues each. These isomeric peptides were prepared by oxidation with cystamine, 2-aminoethanethiolation of residual thiols, and fractionation by reverse-phase high-performance liquid chromatography. Disulfide pairings were established by mapping the tryptic fragments and confirming their composition by amino acid analysis. After protracted incubation under oxidizing conditions at 25.0°C andp H 8.0, the 26-member ring incorporating the native disulfide bond between residues 65 and 72 is the dominant product. Assuming that equilibrium is established, we infer that local interactions in the sequence of ribonuclease A significantly stabilize the native 8-residue disulfide loop with respect to the non-native 8-residue loop (G°=–1.1±0.1 kcal mole^–1). The implications of this observation for the oxidative folding of the intact protein are discussed.     

Protein refolding in reversed micelles

A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant-free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.     

From helix-coil transitions to protein folding

An evolution of procedures to simulate protein structure and folding pathways is described. From an initial focus on the helix-coil transition and on hydrogen-bonding and hydrophobic interactions, our original attempts to determine protein structure and folding pathways were based on an experimental approach. Experiments on the oxidative folding of reduced bovine pancreatic ribonuclease A (RNase A) led to a mechanism by which the molecule folded to the native structure by a minimum of four different pathways. The experiments with RNase A were followed by development of a molecular mechanics approach, first, making use of global optimization procedures and then with molecular dynamics (MD), evolving from an all-atom to a united-residue model. This hierarchical MD approach facilitated probing of the folding trajectory to longer time scales than with all-atom MD, and hence led to the determination of complete folding trajectories, thus far for a protein containing as many as 75 amino acid residues. With increasing refinement of the computational procedures, the computed results are coming closer to experimental observations, providing an understanding as to how physics directs the folding process.