Fragment Of Staphylococcal Nuclease Research Articles

Investigating Interactions Between the Hsp90 Molecular Chaperone and Unfolded Protein Substrates

The ubiquitous protein chaperone Hsp90 plays an integral role in cellular homeostasis and protein folding by interacting with substrate proteins. Only recently have three-dimensional structures of the full-length Hsp90 been determined and a reaction cycle been proposed. Despite this achievement, remarkably little is known about the molecular basis for substrate interactions. To investigate this issue, I am utilizing a well-studied model of an unfolded protein, Δ131Δ, a fragment of staphylococcal nuclease. Using small angle x-ray scattering and structure-based fitting we have found that Hsp90 undergoes an open/closed conformational change in the presence of Δ131Δ. NMR measurements of Δ131Δ and domain fragments of Hsp90 have indicated the interaction location on both the chaperone and the substrate. We use this information to build a mechanistic model of how Hsp90 interacts with unfolded protein substrates and how different nucleotide states influence these interactions.

Triple-resonance NOESY-based experiments with improved spectral resolution: applications to structural characterization of unfolded, partially folded and folded proteins.

NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered states of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform 15N and 13C labeling and make use of the chemical shift dispersion of backbone 15N and 13C' (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C' resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in 1H and aliphatic 13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C' either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), delta 131 delta, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate H alpha protons or protons resonating close to water.

A Comparison of the X-ray Diffraction Patterns of Crystals of Reconstituted Nuclease-T and of Native Staphylococcal Nuclease

Abstract Reconstituted nuclease-T (nuclease-T') composed of two fragments of staphylococcal nuclease containing residues 6 to 48 and residues 49 to 149, respectively, has been crystallized in the presence of deoxythymidine 3',5'-diphosphate and Ca2+. The crystals are tetragonal with space group P41 (or P43) and dimensions of a = b = 47.6 ± 0.2 A, c = 63.3 ± 0.2 A, at 4 to 5° at pH 8.0. These values are very close to those of the liganded nuclease crystal determined by A. Arnone et al. (Arnone, A., Bier, C. J., Cotton, F. A., Hazen, E. E., Jr., Richardson, D. C., and Richardson, J. S. (1969) Proc. Nat. Acad. Sci. U.S.A. 64, 420). The comparison of the (0kl) precession photographs of the crystals of nuclease-T' and of nuclease indicates that these crystals are highly isomorphous. The isomorphism of the crystals of nuclease-T and of nuclease-T' also has been similarly observed. The results are consistent with previous observations showing the close similarity of the three-dimensional structures of nuclease and of nuclease-T (and nuclease-T') in solution.

Semisynthetic Analogues of an Enzymically Active Complex Formed between Two Overlapping Fragments of Staphylococcal Nuclease

Abstract It has been shown previously that a cyanogen bromide fragment of staphylococcal nuclease (149 residues) containing the COOH-terminal portion of the molecule (residues 99 to 149) can undergo noncovalent interaction with a product of limited trypsin digestion of nuclease (residues 1 to 126) to yield a complex with approximately 10% the enzymic activity of the native protein. The two interacting fragments, when alone in solution, appear to be essentially structureless while the active complex exhibits much of the ordered structure of nuclease itself. The studies reported here have investigated, in a qualitative manner, the minimum structural requirements of the (99-149) fragment for productive complementation. The single tryptophan residue of nuclease, at position 140, may be replaced with phenylalanine and the fragment may be shortened at the NH2 terminus to the region of residues 116 to 117 and at the COOH terminus to position 140 without elimination of capacity to support the formation of a complex with at least partial activity.