Kiyonobu Yokota

Comparative analysis of protein thermostability: Differences in amino acid content and substitution at the surfaces and in the core regions of thermophilic and mesophilic proteins

Abstract In order to investigate the factors responsible for protein thermostability, we performed a comparative analysis. For this study, we prepared a new dataset composed of 47 homologous pairs of thermophilic and mesophilic proteins. It is he largest comparative study dataset ever presented. The frequency and substitution preference of each amino acid type in the dataset were analyzed.Twokinds of residual structural states were considered, i.e. surface (solvent-exposed) and core (buried) regions. On the surface of thermophilic proteins, higher frequencies were observed for Arg, Glu, and Tyr. Analysis of substitution preference also suggests that these often appear by replacement of other amino acid types. The results indicate that Arg, Glu, and Tyr are suitable for location on the surface of thermophilic proteins. On the other hand, at the core of thermophilic proteins, Ala is often appeared. In addition, our t-test analysis provides the first quantitative information about trends in the frequencies and substitution preferences for Cys, Gln, Met, and Ser. The results indicate that Gln and Met on the surface and Cys and Ser in the core are disadvantageous for protein thermostability.

A Second Lysine-Specific Serine Protease from Lysobacter sp. Strain IB-9374

A second lysyl endopeptidase gene (lepB) was found immediately upstream of the previously isolated lepA gene encoding a highly active lysyl endopeptidase in Lysobacter genomic DNA. The lepB gene consists of 2,034 nucleotides coding for a protein of 678 amino acids. Amino acid sequence alignment between the lepA and lepB gene products (LepA and LepB) revealed that the LepB precursor protein is composed of a prepeptide (20 amino acids [aa]), a propeptide (184 aa), a mature enzyme (274 aa), and a C-terminal extension peptide (200 aa). The mature enzyme region exhibited 72% sequence identity to its LepA counterpart and conserved all essential amino acids constituting the catalytic triad and the primary determining site for lysine specificity. The lepB gene encoding the propeptide and mature-enzyme portions was overexpressed in Escherichia coli, and the inclusion body produced generated active LepB through appropriate refolding and processing. The purified enzyme, a mature 274-aa lysine-specific endopeptidase, was less active and more sensitive to both temperature and denaturation with urea, guanidine hydrochloride, or sodium dodecyl sulfate than LepA. LepA-based modeling implies that LepB can fold into essentially the same three-dimensional structure as LepA by placing a peptide segment, composed of several inserted amino acids found only in LepB, outside the molecule and that the Tyr169 side chain occupies the site in which the indole ring of Trp169, a built-in modulator for unique peptidase functions of LepA, resides. The results suggest that LepB is an isozyme of LepA and probably has a tertiary structure quite similar to it.