Single deletion analysis and thiol cross -linking of a membrane transport protein: The lactose permease of Escherichia coli

The lactose permease of Escherichia coli is an important model system for secondary active transport proteins. In lieu of a high resolution crystal structure, a battery of biochemical, biophysical, and genetic techniques have been developed in order to obtain both structural and functional information about this protein. This dissertation introduces single deletions as a method for approximating the termini of transmembrane domains of polytopic membrane proteins. The approach is based upon the postulate that alteration of the register of regular transmembrane structures by deletion of single amino acid residues will disrupt helix-helix interactions required for transport activity. In contrast, single deletion of loop residues should be relatively innocuous.;It is demonstrated that 19 of the 24 loop-helix interfaces approximated by loss or diminution of transport activity as the result of deletion of single amino acid residues are within 1 to 4 residues of the termini proposed by hydropathy analysis or other predictive algorithms. Three of the remaining five loop-helix interfaces are consistent with results from site-directed spin labeling studies but differ significantly from prediction. The results suggest that a strong correlation exists between structure and loss of activity as determined by single deletion analysis and support a topological model of the lactose permease consisting of 12 transmembrane alpha-helices with an average length of 21 residues.;A major change in topology suggested by deletion analysis and confirmed by site-directed spin labeling studies is the placement of the Glu126 and Arg144 into helices IV and V, respectively which are the major determinants for substrate binding. Utilizing site-directed thiol-crosslinking, it is shown that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149 which are approximately one helical turn removed from the essential residues, spontaneously cross-link indicating that these residues are in close proximity. Neutralization of Glu126 or both Glu126 and Arg144 with Ala has little effect on the cross-linking of A122C/Val149C. In contrast, neutralization Arg144 with Ala results in marked decrease in cross-linking efficiency, indicating that an unpaired carboxylate at position 126 causes a structural perturbation at the interface between helices IV and V.