Proteolysis Of Native Proteins As A Structural Probe

Abstract

Abstract A common use of proteinases in vitro is the degradation of proteins to limit peptide mixtures in which all susceptible bonds have been cleaved (Chapter 8). To facilitate this process, the substrate would usually be first denatured, by heat, urea, guanidinium hydrochloride, by precipitation with trichloroacetic acid or similar treatments. As a result of the destruction of the higher-order structure in the substrate, all susceptible (i.e. in accordance with the primary cleavage specificity) bonds will be attacked rapidly and with rates that, compared with native state digestions, are approximately similar and influenced only by local changes in subsite occupancy; in this way, the peptide mixture limit is rapidly generated. The need for the prior denaturation step implicitly assumes that retention of native structure will slow or modify the proteolytic attack, and this is indeed the case. The role of three-dimensional structure of a protein substrate in modification of extent and rapidity of proteolysis is incompletely understood, but a recurrent observation is that proteolysis is more limited, both in the extent and in the rate of hydrolysis. This restriction of proteolytic attack on native proteins also underpins most proteolytic interactions of physiological relevance, and thus, the study of limited proteolysis is also relevant to processes in vivo.