| Although we can readily deduce elements of secondary structure from an amino acid sequence, it is still challenging to determine the tertiary structure of a protein. The main challenge, however, is in the determination of the quaternary structures of proteins. In this work, the interactions determining specificity and stability in parallel dimeric coiled-coils were studied.;First, the ability of pyrenes to form excimers was used as a "reporter" to detect the presence of parallel dimeric coiled-coils. The constraints for the formation of excimers were successfully linked to proper folding by appending pyrene molecules to the peptides. This method allows one to rapidly solve the partnering problem (specificity) among peptides believed to form coiled-coils.;Second, a metal binding template was designed for the creation of parallel dimeric coiled-coils. Folding is dependent on metal chelation by a terpyridyl moiety attached to the peptides. The choice of the metal leads to the creation of inert or labile dimers, in which individual peptides can exchange. By fixing the peptide stoichiometry, it becomes possible to specifically look at factors affecting stability in coiled-coils.;Finally, the template was used to generate virtual combinatorial libraries with the ability to self-select the most stable dimers. A substoichiometric amount of metal was used with combinatorial peptide libraries, so that only the most stable dimers would chelate the metal. Dimers and unfolded peptides were separated by Size Exclusion chromatography and analyzed by LC/MS. Interestingly, dimer stability correlates better with peak width than with retention time during Size Exlusion Chromatography. |
