Proteolytic digestion of the micellar complex of f1 coat protein and deoxycholate

Jl Woolford,Re Webster

doi:10.1016/s0021-9258(19)41423-3

Abstract

The major coat protein of bacteriophage f1 radioactively labeled with specific amino acids was solubilized with deoxycholate and digested with trypsin or alpha-chymotrypsin. The degree of proteolysis of the coat protein was assayed by gel filtration chromatography of the digest in the presence of deoxycholate. Hydrolysis occurred at residues in the hydrophilic termini of the coat, releasing peptides containing proline, lysine, and phenylalanine. No cleavage occurred at the tyrosine or methionine residues in the hydrophobic core. However, chymotrypsin could cleave somewhat at these residues in the absence of deoxycholate. A model for the topography of the micellar complex of coat protein and deoxycholate is presented in which the hydrophobic sequence of the coat is bound to deoxycholate within a micelle, while the hydrophilic termini of the coat project from the micelle.

Highlights

In a micellar complex of coat protein and deoxycholate, the hydrophobic region of the coat protein probably resides within the deoxycholate micelle, while the hydrophilic NH2 and COOH termini of the coat probably project into the aqueous solvent outside of the micelle
The fl major coat protein, which is tightly associated with the inner membrane of E. coli during phage maturation (l-3), is a promising model membrane protein, since it can be purified in large quantities, and since its amino acid sequence has been determined [4,5,6,7]
The hydrophobic moiety acts as a nucleus for the formation of micelles with deoxycholate and other detergents [8]. Such binding of detergents has only been found to occur with proteins which occur naturally in association with membranes, such as cytochrome b, [9], the major glycoprotein of the erythrocyte membrane (14, I5), and the membrane glycoproteins of Semliki forest virus [15, 16]

Summary

Introduction

The binding occurs in a cooperative manner and near the cmc, which seems to be common to membrane proteins [9], in contrast to the noncooperative binding of deoxycholate to discrete sites of bovine serum albumin or the total lack of binding of deoxycholate to water-soluble proteins with hydrophilic surfaces [10]. For these reasons, it was suggested that the hydrophobic domain of the coat protein, presumably the detergent-binding site, could act as a nucleus for micelle formation with deoxycholate [8]. ‘The abbreviations used are: Na dodecyl-SO,, sodium dodecyl sulfate; cmc, critical micelle concentration; O.D., optical density; GnHCl, guanidine hydrochloride; TPCK, tosyl-L-phenylalanylchloromethane

Methods

Results

Conclusion