Research Synopsis: Bioactive peptide design and synthesis, protein engineering, ligand-receptor interactions
A major objective of the lab is the development of approaches to understanding the functional conformations of intermediate-sized, flexible peptides with important biological activities. Our basic approach to this problem is the design, synthesis and study of peptide analogues. New methods in solid-phase peptide synthesis are being developed in order to synthesize peptides that are constrained in particular conformations by multiple side-chain to side-chain bridges. For example, lactam bridges linking lysine and aspartic acid residues in i and i+4 positions in the peptide chain are being used to stabilize amphiphilic alpha-helical structures in the peptide hormones beta-endorphin and calcitonin, as well as DNA-binding helical structures such as the basic region from the transcription factor GCN4. More complex structures of this type, simultaneously linking three or four side-chains, are also under development. An important aspect of our strategy is the use of detailed analyses of peptide conformations, by circular dichroism and NMR methods, and of receptor affinities, in order to evaluate the structural and energetic effects of our conformational constraints. In an extension of these studies, we are also investigating helix-stabilized analogues of the transmembrane domains of peptide hormone receptors. The goal of this work is to characterize the helix-helix interactions that define the core of the folded structures of this important class of proteins. Initially, this area of research is focused on the human receptors for opioid peptides such as beta-endorphin, dynorphin and the enkephalins.
Conformationally constrained peptides
A new area in which our research on conformationally constrained peptides has focused is in the design of peptide-based vaccines directed against HIV-1. In this project, we are exploring structure-activity relationships in the binding of an HIV-neutralizing human monoclonal antibody, 2F5, to its recognition epitope on the HIV envelope glycoprotein gp 41. The goal of this project is to develop conformationally constrained analogs of this epitope peptide that bind tightly to 2F5. We are then conjugating these peptides to carrier proteins, so that they can be recognized by the immune system, and we are testing their abilities to elicit an immune response that is (like that of 2F5) HIV neutralizing. These are the initial steps that we hope will lead to potential therapeutic applications in the treatment of HIV-positive patients, and prevention of the development of AIDS.
Properties of peptides at interfaces
In addition to our synthetic approaches, we are also developing model systems for studying the properties of peptides at interfaces. This area of research is being applied in particular to peptide hormones and to peptide segments of serum apolipoproteins that are implicated in either phospholipid binding or in the deposition of low density lipoproteins (LDL) onto damaged arterial walls. The air-water interface of a Langmuir trough and the phospholipid surfaces of single-bilayer vesicles are being used as model interfaces to which such peptides may be bound and studied.
Synthesis and structure of designed polypeptides
The above illustration is of the alpha-helical conformation of a protected synthetic bicyclic peptide, Boc-cyclo(1-5, 2-6)-[Lys-Lys-Ala-Ala-Asp-Asp]-OPac, in a trifluoroethanol/water (1/1) solution. The backbone atoms are traced by a solid green ribbon. Also apparent are the two lactam bridges linking the Lys(1) and Asp(5) side-chains and the Lys(2) and Asp(6) side chains. The molecule is oriented with the N-terminal Boc protecting group at the top, and the C-terminal OPac protecting group at the bottom. These groups can be removed to allow linkage of this peptide to other synthetic peptides for initiation and stabilization of the alpha-helical conformation. The structure of this hexapeptide was determined by 2-D NMR methods in the laboratory of Professor Jean Baum, also in this department, and is described in the 1994 publication by Bracken et al. listed below.