Visual detection of specific, native interactions between soluble and microbead-tethered alpha-helices from membrane proteins

Using peptides tethered to polymer microbeads, we have developed a techniqu e for measuring the interactions between the transmembrane a-helices of mem brane proteins and for screening combinatorial libraries of peptides for me mbers that interact with specific helices from membrane proteins. The metho d was developed using the well-characterized homodimerization sequence of t he membrane-spanning a-helix from the erythrocyte membrane protein glycopho rin A (GPA). As a control.. we also tested a variant with a dimer-disruptin g alteration of a critical glycine residue to leucine. To test for detectab le, native interactions between detergent-solubilized and microbead-tethere d a-helices, we incubated fluorescent dye-labeled GPA analogues in sodium d odecyl sulfate solution with microbeads that contained covalently attached GPA analogues. When the dye-labeled peptide in solution and the bead-tether ed peptide both contained the native glycophorin A sequence, the microbeads readily accumulated the dye through lateral peptide-peptide interactions a nd were visibly fluorescent under UV light. When either the peptide in solu tion or the peptide attached to the beads contained the glycine to leucine change, the beads did not accumulate any dye. The usefulness of this method for screening tethered peptide libraries was tested by incubating dye-labe led, native sequence peptides in detergent solution with a few native seque nce beads plus an excess of beads containing the variant glycine to leucine sequence. When the dye-labeled peptide in solution was present at a concen tration of greater than or equal to2 muM, the few native sequence beads wer e visually distinguishable from the others because of their bright fluoresc ence. Using this model system, we have shown that it is possible to visuall y detect specific, native interactions between a-helices from membrane prot eins using peptides tethered to polymer microbeads. It will thus be possibl e to use this method to measure the specific lateral interactions that driv e the folding and organization of membrane proteins and to screen combinato rial libraries of peptides for members that interact with them.