STRUCTURAL STUDIES OF CONNECTIVE TISSUE

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The involvement of collagens in connective tissue diseases, together with the health related implications of the macrophage scavenger receptor, C1q and other triple-helix containing proteins makes it important to elucidate the properties of the triple-helix. This laboratory has used triple-helical peptides of defined sequence to approach the effect of amino acid sequence on conformation, and our peptides have provided a basis for collaborative studies leading to the first high resolution structure of a triple-helix by x-ray crystallography and the first 2-D NMR studies of triplehelica1 molecules. To understand the propensities of different residues to adopt a triple-helical conformation, a set of host-guest peptides will be synthesized where a single "guest" Gly-X-Y tripeptide unit is introduced into a defined environment of Gly-Pro-Hyp units. The relative stabilities of the most common Gly-X-Y triplets will be applied to detect variations along collagen sequences and will be used, together with molecular modeling, to define interactions stabilizing the triple-helix. The electrostatic interactions of a peptide with a single Gly-Glu-Lys unit have been characterized, and crystals obtained of this peptide will allow a molecular definition of these interactions. Analyses of the frequencies and clustering of tripeptide units will be done on triple-helices of collagens and non-collagenous proteins to clarify features common to all triple-helices and features related to specific higher level interactions. Triple-helical domains interact with a wide variety of ligands, yet there is little information on the molecular interactions which form the basis of specific recognition of sites along the rather uniform triple-helix. To explore the basis of recognition, studies will be carried out on overlapping peptides containing sequences surrounding a collagen epitope to a monoclonal antibody. Experiments are designed to -test the hypothesis that recognition is achieved by the determinant sequence when it is surrounded by flexible triplets containing Gly-Gly sequences. A peptide containing sequences of the triple-helical ligand binding region of the macrophage scavenger receptor will also be studied to determine the basis for its broad yet discriminating polyanionic ligand specificity. Glycine substitutions in type I collagen triple-helices have been found in many cases of osteogenesis imperfecta ("brittle bone" disease), but it is not clear how the variable clinical phenotypes, ranging from mild to lethal, relate to the location of the substitution along the-chain. A single Gly- greater than Ser mutation at residue 901 results in a mild OI case, yet a Gly- greater than Ser at residue 913 results in a lethal OI case. Peptides will be synthesized to clarify whether different local environments of these mutations are the cause of variations seen in collagen stability and clinical phenotype. These studies will make a contribution to understanding the basic interactions required for protein stability as well as developing a structural basis for consideration of the triple-helix, and antibodies to triple-helical domains, in pathological states.