Biomedical subjects

Ervin Welker

Publications and source records attributed to Ervin Welker.

14 recordsLinked to original sources

PubMed2006

A localized specific interaction alters the unfolding pathways of structural homologues.

Reductive unfolding studies of proteins are designed to provide information about intramolecular interactions that govern the formation (and stabilization) of the native state and about folding/unfolding pathways. By mutating Tyr92 to G, A, or L in the model protein, bovine pancreatic ribonuclease A, and through analysis of temperature factors and molecular dynamics simulations of the crystal structures of these mutants, it is demonstrated that the markedly different reductive unfolding rates and pathways of ribonuclease A and its structural homologue onconase can be attributed to a single, localized, ring-stacking interaction between Tyr92 and Pro93 in the bovine variant. The fortuitous location of this specific stabilizing interaction in a disulfide-bond-containing loop region of ribonuclease A results in the localized modulation of protein dynamics that, in turn, enhances the susceptibility of the disulfide bond to reduction leading to an alteration in the reductive unfolding behavior of the homologues. These results have important implications for folding studies involving topological determinants to obtain folding/unfolding rates and pathways, for protein structure-function prediction through fold recognition, and for predicting proteolytic cleavage sites.

Animals↗

PubMed2006

Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry.

Although deamidation at asparagine and glutamine has been found in numerous studies of a variety of proteins, in almost all cases the analytical methodology that was used could detect only a single site of deamidation. For the extensively studied case of reduced bovine ribonuclease A (13,689 Da), only Asn67 deamidation has been demonstrated previously, although one study found three monodeamidated fractions. Here top down tandem mass spectrometry shows that Asn67 deamidation is extensive before Asn71 and Asn94 react; these are more than half deamidated before Asn34 reacts, and its deamidation is extensive before that at Gln74 is initiated. Except for the initial Asn67 site, these large reactivity differences correlate poorly with neighboring amino acid identities and instead indicate residual conformational effects despite the strongly denaturing media that were used; deamidation at Asn67 could enhance that at Asn71, and these enhance that at Gln74. This success in the site-specific quantitation of deamidation in a 14 kDa protein mixture, despite the minimal 1 Da (-NH2 --> -OH) change in the molecular mass, is further evidence of the broad applicability of the top down MS/MS methodology for characterization of protein posttranslational modifications.

Amides↗

PubMed2004

Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A.

The earliest folding events in single-tryptophan mutants of RNase A were investigated by fluorescence measurements by using a combination of stopped-flow and continuous-flow mixing experiments covering the time range from 70 micros to 10 s. An ultrarapid double-jump mixing protocol was used to study refolding from an unfolded ensemble containing only native proline isomers. The continuous-flow measurements revealed a series of kinetic events on the submillisecond time scale that account for the burst-phase signal observed in previous stopped-flow experiments. An initial increase in fluorescence within the 70-micros dead time of the continuous-flow experiment is consistent with a relatively nonspecific collapse of the polypeptide chain whereas a subsequent decrease in fluorescence with a time constant of approximately 80 micros is indicative of a more specific structural event. These rapid conformational changes are not observed if RNase A is allowed to equilibrate under denaturing conditions, resulting in formation of nonnative proline isomers. Thus, contrary to previous expectations, the isomerization state of proline peptide bonds can have a major impact on the structural events during early stages of folding.

Kinetics↗

PubMed2004

Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues.

Using DTT(red) as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT(red). Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30-75) disulfide bond. The reductive unfolding pattern of onconase is consistent with an analysis of the exposed surface area of the cysteine sulfur atoms in the (30-75) disulfide bond, which reveals that these atoms are about four- and sevenfold, respectively, more exposed than those in the next two maximally exposed disulfides. By contrast, in the reductive unfolding of the homologue, RNase A, there are two intermediates, arising from the reduction of the (40-95) and (65-72) disulfide bonds, which takes place in parallel, and on a much longer time-scale, compared to the initial reduction of onconase; this behavior is consistent with the almost equally exposed surface areas of the cysteine sulfur atoms that form the (40-95) and (65-72) disulfide bonds in RNase A and the fourfold more exposed cysteine sulfur atoms of the (30-75) disulfide bond in onconase. Analysis and in silico mutation of the residues around the (40-95) disulfide bond in RNase A, which is analogous to the (30-75) disulfide bond of onconase, reveal that the side-chain of tyrosine 92 of RNase A, a highly conserved residue among mammalian pancreatic ribonucleases, lies atop the (40-95) disulfide bond, resulting in a shielding of the corresponding sulfur atoms from the solvent; such burial of the (30-75) sulfur atoms is absent from onconase, due to the replacement of Tyr92 by Arg73, which is situated away from the (30-75) disulfide bond and into the solvent, resulting in the large exposed surface-area of the cysteine sulfur atoms forming this bond. Removal of Tyr92 from RNase A resulted in the relatively rapid reduction of the mutant to form a single intermediate (des [40-95] Y92A), i.e. it resulted in an onconase-like reductive unfolding behavior. The reduction of the P93A mutant of RNase A proceeds through a single intermediate, the des [40-95] P93A species, as in onconase. Although mutation of Pro93 to Ala does not increase the exposed surface area of the (40-95) cysteine sulfur atoms, structural analysis of the mutant reveals that there is greater flexibility in the (40-95) disulfide bond compared to the (65-72) disulfide bond that may make the (40-95) disulfide bond much easier to expose, consistent with the reductive unfolding pathway and kinetics of P93A. Mutation of Tyr92 to Phe92 in RNase A has no effect on its reductive unfolding pathway, suggesting that the hydrogen bond between the hydroxyl group of Tyr92 and the carbonyl group of Lys37 has no impact on the local unfolding free energy required to expose the (40-95) disulfide bond. Thus, these data shed light on the differences between the reductive unfolding pathways of the two homologous proteins and provide a structural basis for the origin of this difference.

Animals↗

PubMed2004

A new method for rapid characterization of the folding pathways of multidisulfide-containing proteins.

Oxidative folding is a composite process that consists of both the conformational folding to the native three-dimensional structure and the regeneration of the native disulfide bonds of a protein, frequently involving over 100 disulfide intermediate species. Understanding the oxidative folding pathways of a multiple-disulfide-containing protein is a very difficult task that often requires years of devoted research due to the high complexity of the process and the very similar features of the large number of intermediates. Here we developed a method for rapidly delineating the major features of the oxidative folding pathways of a protein. The method examines the temperature dependence of the oxidative folding rate of the protein in combination with reduction pulses. Reduction pulses expose the presence of structured intermediates along the pathways. The correlation between the regeneration rate at different temperatures and the stability of the structured intermediates reveals the role that the intermediates play in determining the pathway. The method was first tested with bovine pancreatic ribonuclease A whose folding pathways were defined earlier. Then, it was explored to discern some of the major features of the folding pathways of its homologue, frog Onconase. The results suggest that the stability of the three-dimensional structure of the native protein is a major determinant of the folding rate in oxidative folding.

Animals↗

PubMed2004

Characterization of the fast-forming intermediate, des [30-75], in the reductive unfolding of onconase.

A fast-forming intermediate in the reductive unfolding of frog onconase (ONC), des [30-75], analogous to the des [40-95] intermediate found in the reductive unfolding of its structural homologue, bovine pancreatic ribonuclease A (RNase A), has been isolated and characterized. The midpoints of the thermal transition and chemical denaturing curves (representing global unfolding) indicate that the conformation of des [30-75] is considerably less stable than that of the parent molecule, suggesting that the (30-75) disulfide bond plays a significant role in the conformational stability of ONC. While des [30-75] is formed very quickly by a partial reduction of the parent molecule in a local unfolding step, it is not as easily susceptible to further reduction, indicating that its three disulfides are much more buried compared to the (30-75) disulfide bond in the parent protein. The nature of des [30-75] is similar to that of des [40-95] RNase A, in that des [30-75] ONC is also a disulfide-secure species. In addition, based on the resistance to mild reducing conditions, structured des species appear to form in ONC from unstructured three-disulfide-containing ensembles. This step is key in the oxidative folding of RNaseA, and is much faster in ONC than the formation of the structured des [40-95] species in RNase A.

Animals↗

PubMed2003

A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates.

The stability of a protein or of its folding intermediates is frequently characterized by its resistance to chemical and/or thermal denaturation. The folding/unfolding process is generally followed by spectroscopic methods such as absorbance, fluorescence, circular dichroism spectroscopy, etc. Here, we demonstrate a new method, by using HPLC, for determining the thermal unfolding transitions of disulfide-containing proteins and their structured folding intermediates. The thermal transitions of a model protein, ribonuclease A (RNase A), and a recently found unfolding intermediate of onconase (ONC), des [30-75], have been estimated by this method. Finally, the advantages of this method over traditional techniques are discussed by providing specific examples.

Chromatography, High Pressure Liquid↗

PubMed2003

Shifting the competition between the intramolecular Reshuffling reaction and the direct oxidation reaction during the oxidative folding of kinetically trapped disulfide-insecure intermediates.

The oxidative folding pathway(s) of single-domain proteins can be characterized by the existence, stability, and structural nature of the intermediates that populate the regeneration pathway. Structured intermediates can be disulfide-secure in that they are able to protect their existing (native) disulfide bonds from SH/SS reshuffling and reduction reactions, and thereby form the native protein directly, i.e., by oxidation of their exposed (or locally exposable) thiols. Alternatively, they can be disulfide-insecure, usually requiring global unfolding to expose their free thiols. However, such an unfolding event also exposes the existing native disulfide bonds. Thus, the subsequent oxidation reaction to form the native protein in a disulfide-insecure intermediate competes with the intramolecular attack by the thiols of the macromolecule on its own native disulfide bonds, resulting in a large population of intermediates that are reshuffled instead of being oxidized. Under stabilizing conditions, disulfide-insecure species become long-lived kinetically trapped intermediates that slowly and only indirectly convert to the native protein through reshuffling reactions. In this study, trans-[Pt(en)(2)Cl(2)](2+), a strong oxidizing agent which has not traditionally been used in oxidative folding, was applied to shift the competition between reshuffling and oxidation reactions in des [58-110] and des [26-84], two long-lived disulfide-insecure intermediates of RNase A, and oxidize them directly under stable conditions to form the native protein. Such a successful direct conversion of kinetically trapped intermediates to the native molecule by trans-[Pt(en)(2)Cl(2)](2+) may be helpful in facilitating the oxidative folding processes of multi-disulfide-containing proteins in general.

Animals↗

PubMed2003

Characterizing the unstructured intermediates in oxidative folding.

A recently developed method is used here to characterize some of the folding intermediates, and the oxidative folding processes, of RNase A. This method is based on the ability of trans-[Pt(en)(2)Cl(2)](2+) to oxidize cysteine residues to form disulfide bonds faster than the disulfide bonds can be rearranged by reshuffling or reduction. Variations of this method have enabled us to address three issues. (i) How the nature of the residual structure and/or conformational order that is present, or develops, during the initial stages of folding can be elucidated. It is shown here that there is a 10-fold increase in the propensity of the unfolded reduced forms of RNase A to form the native set of disulfides directly, compared to the propensity under strongly denaturing conditions (4-6 M GdnHCl). Thus, the unfolded reduced forms of RNase A are not statistical coils with a more condensed form than in the GdnHCl-denatured state; rather, it is suggested that reduced RNase A has a little bias toward a native topology. (ii) The structural characterization of oxidative folding intermediates in terms of disulfide pairing is demonstrated; specifically, a lower-limit estimate is made of the percentage of native disulfide-containing molecules in the two-disulfide ensemble of RNase A. (iii) The critical role of structured intermediate species in determining the oxidative folding pathways of proteins was shown previously. Here, we demonstrate that the presence of a structured intermediate in the oxidative folding of proteins can be revealed by this method.

Chromatography, High Pressure Liquid↗

PubMed2003

Native conformational tendencies in unfolded polypeptides: development of a novel method to assess native conformational tendencies in the reduced forms of multiple disulfide-bonded proteins.

Oxidative folding is the concomitant formation of the native disulfide bonds and the native tertiary structure from the reduced and unfolded polypeptide. Of interest is the inherent conformational tendency (bias) present in the reduced polypeptide to dictate the formation of the full set of native disulfide bonds. Here, by application of a novel tool, we have been able to assess this "native conformational tendency" present in reduced and unfolded bovine pancreatic ribonuclease A (RNase A). The essence of this method lies in the ability of the oxidant [Pt(en)(2)Cl(2)](2+) (where "en" is ethylenediamine) to oxidize disulfide bonds under conditions in which both reduction and disulfide reshuffling, which are essential for rearranging non-native disulfide bonds, are extremely slow. When applied to RNase A, the method revealed little or no bias toward formation of the full native set of disulfide bonds in the fully reduced protein.

Animals↗

PubMed2002

Proline cis-trans isomerization and protein folding.

Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding. The energetic origin of this isomerization process is summarized, and the folding and unfolding of disulfide-intact bovine pancreatic ribonuclease A is used as an example to illustrate the kinetics and structural features of conformational changes from the heterogeneous unfolded state (consisting of cis and trans isomers of X-Pro peptide groups) to the native structure in which only one set of proline isomers is present.

Animals↗

PubMed2002

Intramolecular versus intermolecular disulfide bonds in prion proteins.

Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.

Animals↗

PubMed2002

Development of a novel method to populate native disulfide-bonded intermediates for structural characterization of proteins: implications for the mechanism of oxidative folding of RNase A.

RNase A, a model protein for oxidative folding studies, has four native disulfide bonds. The roles of des [40-95] and des [65-72], the two native-like structured three-disulfide-bonded intermediates populated between 8 and 25 degrees C during the oxidative folding of RNase A, are well characterized. Recent work focuses on both the formation of these structured disulfide intermediates from their unstructured precursors and on the subsequent oxidation of the structured species to form the native protein. The major obstacles in this work are the very low concentration of the precursor species and the difficulty of isolating some of the structured intermediates. Here, we demonstrate a novel method that enables the native disulfide-bonded intermediates to be populated and studied regardless of whether they have stable structure and/or are present at low concentrations during the oxidative folding or reductive unfolding process. The application of this method enabled us to populate and, in turn, study the key intermediates with two native disulfide bonds on the oxidative folding pathway of RNase A; it also facilitated the isolation of des [58-110] and des [26-84], the other two native-like structured des species whose isolation had thus far not been possible.

Animals↗

PubMed2002

Conformational propensities of protein folding intermediates: distribution of species in the 1S, 2S, and 3S ensembles of the [C40A,C95A] mutant of bovine pancreatic ribonuclease A.

A key problem in experimental protein folding is that of characterizing the conformational ensemble of denatured proteins under folding conditions. We address this problem by studying the conformational propensities of reductively unfolded RNase A under folding conditions, since earlier work has indicated that the equilibrium conformational ensemble of fully reduced RNase A resembles the transient conformational ensemble of a burst-phase folding intermediate of disulfide-intact RNase A. To assess these propensities, the relative disulfide-bond populations of the 1S, 2S, and 3S ensembles of the [C40A,C95A] mutant of RNase A were measured. Thirteen of the fifteen possible disulfide bonds are observed, consistent with earlier results and with the rapid reshuffling and lack of stable tertiary structure in these ensembles. This broad distribution contradicts recent observations by another group, but rigorous cross-checks show unambiguously that our data are self-consistent whereas their data are not. The distributions of disulfide bonds in the wild-type and mutant proteins show a power-law dependence on loop length, with an exponent that is significantly smaller than the exponents of either ideal or excluded-volume polymers. The 65-72 disulfide bond is much more strongly favored than would be predicted by this power law, consistent with earlier peptide studies and the disulfide-bond distributions of the 1S and 2S ensembles in wild-type RNase A. Experimental evidence suggests that this preference results from conformational biases in the backbone, rather than from differing accessibilities or reactivities of the two cysteine residues. In general, the other disulfide species do not deviate significantly from the power-law dependence, indicating that the conformational biases are relatively weak.

Alanine↗