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Structural Characterization of a Streptococcus Pyogenes collagen-like protein and its usefulness in determining collagenase specificity

Zhuoxiun Yu
B.S., Nankai University- 2007

Thesis Advisors: Masayori Inouye, Ph.D., and Barbara Brodsky, Ph.D.

Graduate Program Biochemistry & Molecular Biology

CABM, Room 010

Monday, December 12, 2011
2:00 p.m.


Even though collagen is considered as an animal protein, more than a hundred collagen-like sequences with Gly as every third residue have been found in bacterial genomes. Several bacterial collagen-like proteins have been expressed and observed to form stable triple-helical structures despite the absence of hydroxyproline. The goal of my thesis research study is to characterize the biochemical and biophysical properties of bacterial collagen-like proteins and to utilize this bacterial collagen system to characterize the degradation of collagen by collagenases. The high yield cold-shock expression system was used to obtain recombinant Scl2 collagen-like protein from Streptococcus pyogenes containing an N-terminal globular domain V followed by the collagen triple-helix domain CL. The V-CL protein forms a triple-helix with a sharp thermal transition at 35-37 oC. The triple-helix domain has the potential of forming some fibrillar structures. The trimerization V domain is required for V-CL folding, which can also fold and solubilize a heterologous triple-helix domain from Clostridium perfringens.

To investigate the relationship between sequence, stability, and folding, the collagenous domain (Gly-Xaa-Yaa)79 sequence was divided to create three subdomains A, B, and C of almost equal size with distinctive amino acid features. Each subdomain was fused with V domain as a monomer (such as V-A) , a dimer (V-AA) and a trimer (V-AAA). The stability and folding rates of these proteins varied depending on the sequence. With increasing triple helix length, the stability was higher and the folding rate was slower suggesting propagation is a rate limiting factor.

This bacterial collagen system was also used to study collagen degradation by collagenases. Sequences surrounding the collagenase cleavage site in human type III collagen were inserted between two CL domains of the V-CL-CL protein. The minimal sequence necessary for collagenase cleavage was shown to include one triplet prior to the G~IA cleavable unit, and three triplets following it. The introduction of an effective collagenase cleavage sequence also leads to trypsin susceptibility, suggesting a locally unfolded state. The recombinant chimeric collagen molecules studied here serve as a good model to gain information on collagenase specificity and mechanism.

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