Solubilization and functional analysis of the lambda holin

Abstract

The 105aa lambda S protein is the prototype holin, S accumulates in the cytoplasmic membrane during late gene expression until, at a time programmed into its primary structure, it disrupts the membrane and allows the lambda lysozyme, R, to attack the cell wall. In this study, a zwitterionic detergent Empigen BB, was used to extract and purify the lambda holin S. In Empigen BB, CD analysis on S gave 54% alpha helical content, consistent with 3 TM domains, which has been reported by other in vivo studies. Empigen BB-purified S can be exchanged into a chaotropic solution by dialysis and reconstituted into preformed lipid vesicles for activity assays. When diluted to fluorescein-loaded suspensions of liposomes, different chaotrope-solubilized S alleles caused dye release reflective of their in vivo phenotypes. The problem was the low efficiency of delivery of S to the liposomes. Unfortunately, dye loaded liposomes are highly sensitive to any detergent, making it necessary to find other ways to solubilize S. GroEL, a chaperonin from E. coli, is responsible for folding and refolding globular proteins in vitro. It has also been reported that GroEL improves the ability of a membrane protein synthesized in vitro to insert post-translationally into liposomes. This work will investigate the behavior of GroEL towards membrane proteins. The first of two membrane proteins studied in this respect is Bacteriorhodopsin (BR), a membrane proton pump, from H. halibium. The second is the105aa S protein, a prototype holin from bacteriophage lambda. Holin and BR subjected to detergent removal in the presence of GroEL remained in solution, while in the control sample (without GroEL) S and BR precipitated. "GroELsolubilized" holin still retained its lesion forming activity and solubilized BR maintained its proton pumping ability, detected by using a liposome dye activity assay unique to each protein. This approach may be applicable to other systems requiring detergent- or chaotrope-free preparation of membrane proteins. Finally, these results suggest that GroEL may be involved in the insertion of integral membrane proteins into the lipid bilayer, a role heretofore unsuspected.