Domain characteristics of the carboxyl-terminal fragment 206-316 of thermolysin: immunochemical studies

Abstract

The extent of nativeness of the stable conformation of the [Bacillus thermoproteolyticus] thermolysin fragment containing the carboxyl-terminal third of the protein (from residues 206 to 316, denoted fragment FII) was examined by its immunogenic and antigenic characteristics. Antisera elicited in rabbits by either intact thermolysin or fragment FII were fractionated serially on 2 affinity columns, containing either the isolated fragment or intact protein. Both sera gave rise to substantial antibody populations which recognized the fragment FII region in native thermolysin. The relative affinities of these specific antibodies for isolated fragment FII and intact thermolysin were evaluated by radioimmunoassay, by assessing the relative extents of competition by these for binding of 14C-labeled thermolysin or 14C-labeled fragment FII to each antibody population. Competition by fragment FII was substantial, though generally weaker than that for intact thermolysin, for antibody binding of both labeled antigens. The stable structure of fragment FII as observed spectroscopically is probably one which possesses conformational features similar to those of this region in intact thermolysin, but with perhaps less conformational rigidity. The region of thermolysin composed primarily of residues 206-316 is apparently a conformational domain of the intact protein. Isolated fragment FII apparently retains domain-like characteristics of stable and native-like conformation.