Volume 2, Number 3/4, p. 101-107
A. Colomer-Pallas, M.-F. Petit-Glatron and R. Chambert
Institut Jacques Monod, Laboratoire GÈnÈtique et Membranes,Centre National de la Recherche Scientifique, UniversitÈs Paris 6 et Paris 7, France
e-mail: colomer-pallas@ijm.jussieu.fr; glatron@ccr.jussieu.fr; chambert@ccr.jussieu.fr
In vitro analysis of aggregation-disaggregation of the folding intermediate of Bacillus subtilis a-amylase
The refolding intermediate of Bacillus subtilis a-amylase is prone to aggregate at 37 ƒC and pH 7 when the protein concentration is relatively high ( „ 1 mM). Low concentrations of 2,2,2 trifluoroethanol greatly increased the rate of aggregation. Aggregation made the folding intermediate resistant to proteases and there was kinetic competition between aggregation and proteolytic degradation. Analysis by Fourier transform infrared spectroscopy indicated that the secondary structure of the refolding intermediate is slightly different under soluble or aggregated states. Aggregates were readily solubilized by guanidium chloride (D1/2 = 1.25 M), but disaggregation was slow when aggregates were resuspended in solutions of various foreign native proteins under physiological conditions of pH and temperature. This destabilizing effect resulting from protein-protein interactions may make the aggregation process reversible in vivo.