H n.m.r. spectrum from the flexible N‐terminal segment of Streptomyces subtilisin inhibitor

Abstract

The 'H n.m.r. spectrum of Streptomyces subtilisin inhibitor shows a limited number of unusually sharp signals at room temperature. Some of these signals are assigned uniquely to protons of the side chains of the N-terminal segment, Aspl-Ala2-Pro3-Ser4-AlaS-Leu6-Tyr7-based on experiments of spin decoupling, pH titration, and enzymatic cleavage of the protein. Quantitative examination of these signals indicates that the N-terminal end of this protein is heterogeneous in that the protein contains a considerable fraction whose sequence starts with Ala2 rather than with Aspl. The pK, values for the amino groups of Aspl and Ala2 exposed at the N-terminus are determined to be 8.9 * 0.4 and 9.0 * 0.1, respectively. Furthermore, examination of the line-widths of the methyl proton resonances of Ala2 and Alas residues indicates that the N-terminal peptide segment is free and undergoes rapid segmental motions in the order of lo-' s.

The N‐terminal peptide of the transglutaminase‐activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross‐linking sites

“…Former NMR studies have shown flexibility of the N‐terminal peptide of SSI from S. albogriseolus . Despite a low similarity, the extension peptide of SSTI from S. mobaraensis , upstream from the LY motif, shares at least four amino acids with the SSI N terminus ( cf .…”

“…Noticeably, the complex formation of 3aa‐ r SSTI (+AQQ) and 6aa‐ r SSTI (+APAAQQ) with TAMP yielded in a positive or, at least, considerably less negative entropy as compared to the wild‐type or the N‐terminally shortened proteins. We assume that the hydrophobic extension peptide is highly flexible and incapable of interacting with the core protein of SSTI . The observed increase in entropy may then result from an energy‐rich interface between water and the hydrophobic peptide that is abolished by the attachment of TAMP.…”

“…Moreover, although solvent accessibility has been assigned to all SSI tyrosines, and, in the crystal structure, SSI‐Tyr106 (SSTI‐Tyr110) seemed to be more exposed than SSI‐Tyr38 (SSTI‐Tyr41) , solvation of SSI‐Tyr106 was dependent on increasing temperatures, and SSI‐Tyr38 was more easily modified by tetranitromethane . A 1 H‐NMR study noted in addition that the N‐terminal peptide of SSI is free and undergoes rapid motions on the nanosecond timescale . Such flexibility explains why we and others could not determine the structure of the N terminus and even not of the fused SSTI extension peptide.…”

“…Although SSTI was extensively modified via point mutations at the N-terminal peptide and the already determined binding sites for the metalloprotease ScNP and the serine proteases subtilisin/trypsin/chymotrypsin, clear results were only obtained with variants exhibiting extended or truncated SSTI N termini. We further crystallized SSTI to gain deeper insights into the MTG-accessible glutamines and the molecular structure of the TAMP-binding site, even though the N-terminal peptide of SSI is highly flexible [14]. Here, we report the protein interactions between SSTI/MTG and SSTI/TAMP as observed by kinetic and thermodynamic measurements and illuminate the respective binding sites in the structure of SSTI.…”