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theses

Triple helical collagen in the most abundant protein found in the human body. It is made of three right-handed polyproline chains that supercoil together with an n-1 stagger to produce a rigid left-handed helix. Collagen has various types based on the combination of chains used to assemble the helix and each type can generate various higher order structures, such as a thick collagen fibril. Collagen’s unique G-X-Y repeated sequence and interstrand bonding network contributes to the protein’s high overall structural integrity, with the GPO triplet (GPO) being the most rigid structure and, therefore, the most often repeated sequence. A subdomain in the collagen sequence with greater internal motion and flexibility is known to be areas of molecular recognition for proteins such as the transmembrane protein integrin or the MHC-I protein subunit β2-microglobulin (B2m). As full length collagen proteins are approximately 1000 residues in length, collagen model peptides of thirty residues are often used for characterization experiments in nuclear magnetic resonance (NMR) but are not necessary in indirect ELISA binding assays. NMR relaxation experiments were conducted on a homotrimeric collagen model peptide GFOGER, designed from the high affinity integrin binding sequence in heterotrimeric type I collagen. Previous research indicated key areas of binding between the two proteins, but flexibility information about the six-residue sequence is essential for future binding studies with heterotrimer models. Experiments with longitudinal relaxation and multiple transverse relaxation experiments have indicated the flexible nature of the insert, and further work will be conducted to identify individual trimer residues and their roles in integrin binding. Indirect ELISA binding assays were conducted between full length type I collagen and B2m to first confirm reports from previous studies, and to work toward potentially identifying the binding domains on collagen that result in the initial binding and subsequent aggregation of B2m. Work in replicating the binding assays did confirm a strong binding interaction between collagen and B2m by experimenting with constant collagen concentrations and various B2m concentrations, ranging from the normal serum level to levels that are what is found in patients undergoing hemodialysis attributed to renal failure.

ETD_5127

M.S.

Includes bibliographical references

by Robert T. Young

doi:10.7282/T3B27SBJ

ETD graduate

The author owns the copyright to this work.