Michael W. Pantoliano

Crystal structure and ligand-binding studies of a screened peptide complexed with streptavidin.

The thermodynamic binding parameters and crystal structure for streptavidin-peptide complexes where the peptide sequences were obtained by random screening methods are reported. The affinities between streptavidin and two heptapeptides were determined by titrating calorimetric methods [Phe-Ser-His-Pro-Gln-Asn-Thr, Ka = 7944 (+/- 224) M-1, delta G degrees = -5.32 (+/- 0.01) kcal/mol, and delta H degrees = -19.34 (+/- 0.48) kcal/mol; His-Asp-His-Pro-Gln-Asn-Leu, Ka = 3542 (+/- 146) M-1, delta G degrees = -4.84 (+/- 0.03) kcal/mol, and delta H degrees = -19.00 (+/- 0.64) kcal/mol]. The crystal structure of streptavidin complexed with one of these peptides has been determined at 2.0-A resolution. The peptide (Phe-Ser-His-Pro-Gln-Asn-Thr) binds in a turn conformation with the histidine, proline, and glutamine side chains oriented inward at the biotin-binding site. A water molecule is immobilized between the histidine and glutamine side chains of the peptide and an aspartic acid side chain of the protein. Although some of the residues that participate in binding biotin also interact with the screened peptide, the peptide adopts an alternate method of utilizing binding determinants in the biotin-binding site of streptavidin.

A pH-dependent superoxide dismutase activity for zinc-free bovine erythrocuprein. Reexamination of the role of zinc in the holoprotein.

The zinc-free derivative of bovine erythrocuprein, Cu2E2BE, was prepared and its superoxide dismutase activity was measured and compared with that of the holoprotein, Cu2Zn2BE. The dismutase activity of these proteins was measured by quantitating their inhibition of the superoxide-mediated autooxidation of 6-hydroxydopamine, dihydroxyfumaric acid, pyrogallol, and epinephrine. It was found that the superoxide dismutase activity of the zinc-free protein is pH dependent, ranging between 82 +/- 5% (relative to Cu2Zn2BE) at pH 5.8, and 25 +/- 10% at pH 10.2. The overlapping range of assays and buffers verified that these measurements are independent of the method of assay, buffer, and ionic strength (in the range of mu = 0.10 to 0.20). The variation in activity with pH is probably due, at least in part, to the migration of Cu(II) at high pH as described previously [J. S. Valentine, M. W. Pantoliano, P. J. McDonnell, A. R. Burger, and S. J. Lippard, Proc. Natl. Acad. Sci. USA 76, 4245 (1979)], since Cu(II) bound at the zinc binding site has been shown to have little or no dismutase activity. The observation of high activity (82%) for the zinc-free protein at pH 5.8, where Cu(II) is predominantly in the native Cu binding site, and less susceptible to removal by ethylenediaminetetraacetic acid, demonstrates that the presence of Zn(II) in Cu2Zn2BE does not greatly enhance the inherent dismutase activity of Cu(II) in the holoprotein.

Engineering a Stable Protease

We have used several approaches to engineer large increases in the stability of the Bacillus serine protease, subtilisin. These include introducing disulfide cross-links, improving electrostatic interactions at calcium ion binding sites, and the use of in vitro random mutagenesis coupled with a phenotypic screen to identify stabilizing mutational events. More than twenty individual stabilizing mutations of subtilisin BPN’ have been identified. Thermodynamic analysis has shown that individually these modifications contribute between 0.3–1.5 Kcal/mol to the free energy of stabilization. We have further found that combining individual stabilizing mutations results in cumulative increases in stability. Calorimetric and crystallographic data demonstrate that increases in the free energy of stabilization are often independent and additive. We therefore have been able to create extremely stable versions of subtilisins in a step by step manner. Thermodynamic stability of subtilisin was also shown to be related to resistance to irreversible inactivation at high temperature and high pH. The most stable versions have half-lives at high pH or high temperature approaching 1000-times longer than the wild type subtilisin BPN’.