IDENTIFICATION OF LOCAL CARBOXY-TERMINAL HYDROPHOBIC INTERACTIONS ESSENTIAL FOR FOLDING OR STABILITY OF CHLORAMPHENICOL ACETYLTRANSFERASE

The role of the carboxy terminal in folding and stabilization of type I chloramphenicol acetyltransferase (CAT(I)) has been studied by mutag enesis and Fourier transform infrared analysis. We have shown that a C AT mutant truncated by seven amino acid residues folds into active pro tein. In this study, the last three residues of this truncated CAT mut ant were randomized to detect structural information required for achi eving a native enzyme conformation. Statistical analysis of sequencing data from randomly chosen mutants revealed that the amino-terminal CA T fragment of 212 amino acid residues is the shortest deletion mutant able to adopt a soluble, enzymatically active structure. This minimal length corresponds to a protein with full-length alpha(5)-helix in the three-dimensional crystal structure of CAT type III. The amino acid p references at the carboxy terminal in the randomization experiments su ggest that this helix also forms completely in the shortened CAT mutan ts. In addition correct folding and/or stabilization requires the form ation of a hydrophobic microdomain at the end of the alpha(5)-helix. T he role of this hydrophobic interaction in CAT folding and structure s tabilization is discussed. (C) 1996 Academic Press Limited