Folding of thermolysin fragments

Sedimentation analysis in the analytical ultracentrifuge has been used to characterize the size and shape of thermolysin and a number of its fragments obtained by chemical or enzymatic cleavage of the protein. Four fragments (121–316, 206–316, 225/226–316 and 255–316) originate from the C‐terminal domain, and two (1–155 and 1–205) from the N‐terminal domain of the intact molecule. In aqueous solution at neutral pH the hydrodynamic properties of the C‐terminal fragments, except 255–316, are consistent with compact homogeneous monomers. Fragment 255–316 is a monomeric species below 0.08 mg/ml concentration and forms a dimer above this concentration. Dimerization does not lead to changes in fragment conformation, as determined by farultraviolet circular dichroic measurements, but to an increase of 5.6°C (to 68.2°C at 1.0 mg/ml) in the temperature for thermal unfolding and a corresponding increase of 4.6 kJ/mol in the free energy of unfolding. Fragments derived from the N‐terminal domain show a strong tendency to form high‐molecular‐mass aggregates. Previous experiments utilizing circular dichroic measurements and antibody binding data suggested that the C‐terminal fragments listed above are able to refold in aqueous solution at neutral pH into a stable conformation of native‐like characteristics [Dalzoppo, D., Vita, C. & Fontana, A. (1985) J. Mol. Biol. 182 , 331–340] (and references cited therein). Present data establish that all these C‐terminal fragments are globular monomeric species in solution (at concentrations ∼ 0.1 mg/ml) and thus represent ‘isolated’ domains (or subdomains) with intrinsic conformational stability typical of small globular proteins.