Dataset Information

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease.

ABSTRACT: Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease (SNase110) have been studied by various biophysical and NMR methods. Samples of G-88W- and V-66W-mutant SNase110, namely G-88W110 and V-66W110, in aqueous solution and SNase110 in 2.0 M TMAO are adopted in this study. The unfolding transitions and folded conformations of the three SNase fragments were detected by far- and near-ultraviolet circular dichroism and intrinsic tryptophan fluorescence measurements. The tertiary structures and internal motions of the fragments were determined by NMR spectroscopy. Both G-88W and V-66W single mutations as well as a small organic osmolyte (Trimethylamine N-oxide, TMAO) can fold the fragment into a native-like conformation. However, the tertiary structures of the three fragments exhibit different degrees of folding stability and compactness. G-88W110 adopts a relatively rigid structure representing a most stable native-like beta-subdomain conformation of the three fragments. V-66W110- and TMAO-stabilized SNase110 produce less compact structures having a less stable "beta-barrel" structural region. The different folding status accounts for the different backbone dynamic and urea-unfolding transition features of the three fragments. The G-20I/G-29I-mutant variants of the three fragments have provided the evidence that the folding status is correlated closely to the packing of the beta-strands in the beta-barrel of the fragments. The native-like beta-barrel structural region acts as a nonlocal nucleus for folding the fragment. The tertiary folding of the three fragments is initiated by formation of the local nucleation sites at two beta-turn regions, I-18-D-21 and Y-27-Q-30, and developed by the formation of a nonlocal nucleation site at the beta-barrel region. The formation of beta-barrel and overall structure is concerted, but the level of cooperativity is different for the three 1-110 residues SNase fragments.

SUBMITTER: Xie T

PROVIDER: S-EPMC1861773 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease.

Xie Tao T Liu Dongsheng D Feng Yingang Y Shan Lu L Wang Jinfeng J

Biophysical journal 20061215 6

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease (SNase110) have been studied by various biophysical and NMR methods. Samples of G-88W- and V-66W-mutant SNase110, namely G-88W110 and V-66W110, in aqueous solution and SNase110 in 2.0 M TMAO are adopted in this study. The unfolding transitions and folded conformations of the three SNase fragments were detected by far- and near-ultraviolet circular dichroism and intrinsic tryptophan fluore ...[more]

PMID: 17172296

Similar Datasets

Project description:Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.

Project description:To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 micros to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the beta-barrel and the N-terminal part of the alpha-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.

Project description:The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.

Project description:To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (Delta G(HX)) and the kinetic unfolding and refolding rates (k(op) and k(cl)) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 x 10(-6) s(-1) and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the beta-barrel, including mutually H-bonding residues in the beta 4 and beta 5 strands, a part of the beta 3 strand that H-bonds to beta 5, and residues at the N-terminus of the alpha2 helix that is capped by beta 5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native alpha2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded beta1-beta2-beta 3 meander, completing the native beta-barrel, plus an adjacent part of the alpha1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.

Dataset Information

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease.

Publications

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets