PROTEIN ENGINEERING - PEPTIDE HORMONE DESIGN

The problem of understanding the functional conformations of flexible, intermediate-sized, bioactive peptides is to be addressed. The hypothesis that these peptides will often have amphiphilic secondary structures induced in them when they express their activities at biological interfaces will be tested. Neuropeptide Y (NPY), calcitonin and calcitonin gene related peptide (CGRP) will serve as examples where there is already good evidence that amphiphilic alpha-helical structures are functionally important for different reasons. New synthetic methodology will be employed to design analogues of NPY that incorporate one or more helix-stabilizing side-chain to side-chain crosslinks in the appropriate positions, and an optimal helix-stabilizing structure will be developed. The best crosslink structure will then also be incorporated into multiple positions along the proposed amphiphilic helical segments of calcitonin and CGRP, using a solid-phase fragment condensation approach to the syntheses. The solution conformations, helix stability, and conformation induction in these peptides at model interfaces will be examined, and then these physicochemical properties will be compared to their pharmacological potencies and pharmacokinetic behavior in an attempt to establish predictive relationships. Determining the positions and functional importance of the amphiphilic helical segments will then allow these peptides to be considered in terms of 3-4 structural domains having distinctly different characteristics, including beta turns, an extended beta strands, flexible hydrophilic linkers, and a polyproline Il-like helix that is also amphiphilic. These extended structural hypotheses will be investigated through the continued study of analogues incorporating suitable model structures and mimetics. Ultimately, it is hoped that the spatial relationship of these separate domains will be established through the introduction of suitable conformational constraints and crosslinks, thereby establishing the complete active conformations. The methods of design, synthesis and physicochemical characterization to be developed in this project will be generally applicable to the problem of understanding functional conformations in large, flexible peptides, and to protein engineering studies of even broader scope.