Folding of thermolysin fragments

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Abstract
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.

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