Alteration of the internal dynamics of Escherichia coli alkaline phosphatase under proteolytic modification, thermal inactivation and refolding

Abstract

The internal dynamics of proteins plays an important role in regulation and realization their functional activity [1–4]. In recent years the room temperature phosphorescence properties of Trp residues have been shown to be a sensitive monitor of the internal dynamics of the surrounding protein matrix [3–7]. In this work we take advantage of the exclusive sensitivity of the room temperature tryptophan phosphorescence (RTTP) lifetime to the local flexibility of the protein environment of the emitting Trp residues to detect the changes in the dynamical structure of Escherichia coli alkaline phosphatase (AP) in solution that is brought about by trypsin proteolytic modification, upon inactivation of the protein at 90°C and the following recovery of its enzyme activity after high temperature action.