Mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein...

Methods for mapping endpoints of partial proteolysis fragments from regulatory subunit of type I cyclic AMP-dependent protein kinase are described with a view to using such data for fine-structure analysis of mutations and/or modifications affecting the protein's electrostatic charge. Peptides generated from [35S]methionine-labeled regulatory subunit were separated by high-resolution two-dimensional gel electrophoresis. Sites of papain cleavage in denatured regulatory subunit were deduced from the kinetics of the appearance, molecular weights, and relative isoelectric points of the fragments produced. These sites and sites of chymotrypsin digestion in the native protein were confirmed by studying peptide overlaps. Carboxy-terminal peptides were identified both by overlaps with cyclic AMP-binding chymotryptic fragments and by their preferential labeling during polysome runoff mediated by pactamycin, an inhibitor of protein initiation. Since peptides containing modifications or mutations that alter protein charge can be identified by shifts in first-dimension isoelectric focusing gel positions, knowledge of fragment endpoints will permit rapid mapping of sites of such alterations by two-dimensional gel analysis of partial proteolytic digests. Such a mapping procedure is inexpensive, can be applied to partially purified proteins or to proteins eluted from polyacrylamide gels, requires only nanogram amounts of the protein of interest, and does not require sequence data to determine relative positions of peptides. Therefore, it provides an attractive alternative to more classical peptide analysis for studying point mutations in cellular proteins of low abundance.