Digestion of the lambda cI Repressor with Various Serine Proteases and Correlation with Its Three Dimensional Structure

Abstract

Partial proteolysis of the lambda cl repressor has been carried Out systematically with trypsin. chymortrypsin, elastase, endoproteinase Glu-C, kallikrein, and thrombin. The cleavage sites have been determined by (i) comparison of fragments produced and observed in SDS-poly-acrylamide gel with known fragments and plots of distance migrated versus log (molecular weight of fragment), (ii) partial Edman sequencing of the stable C-terminal fragments to identify cleavage points, and (iii) electrospray mass spectrometry of fragments produced. Most cleavage points are found to occur in the region 86-137, saving some in the N-terminal domain observed for trypsin and Glu-C. Region 86-137 can be further subdivided into three regions 86-91, 114-121, and 128-137 prone to cleavage. with intermediate regions resistant to cleavage to all six proteases. These resistant regions show that Much of the region 93-131 previously called a 'linker' is actually part of the C-domain its first proposed in all models from our laboratory. Region 92-114 includes the cleavage site Ala-Gly, which Must be buried in the intact repressor. The observed cleavage points in region 114-137 call be used to judge the best among three previously proposed models since they differ front each other in the structure of region 93-131. Model lj5g, is adjudged to be better than model 11wq (which is based oil 1kca. a crystal structure) as Susceptible residues are more exposed in the former and lack of cleavages at six sites is better explained. Likewise, the models lj5j and 11wq are compared with a recent crystal Structure of fragment 101-229 in 2ho0 and another low resolution crystal structure in 3bdn.