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Natural interruptions and Gly missense mutations in type IV collagen: modeling effects on structure, folding, and function

Eileen S. Hwang
B.A., Princeton University - 2005

Thesis Advisor: Barbara Brodsky, Ph.D.
Graduate Program in Biochemistry & Molecular Biology

6th Floor Conference Room
RWJMS Research Tower

Tuesday, June 21, 2011
10:00 a.m.


Non-fibrillar collagens contain interruptions in the repeating amino acid sequence pattern, (Gly-Xaa-Yaa)n, that is required for the standard collagen triple helix. Type IV collagen contains more than 20 interruption sites of various lengths, yet a missense mutation replacing one Gly by a larger residue results in Alport syndrome, an inherited kidney disease. The goal of my thesis research study is to clarify why natural interruptions are tolerated while Gly missense mutations lead to a pathological condition. I examined the structural consequences of natural interruptions, and their relation to disease causing mutations, using computational analysis, model peptides, and a recombinant system.

A computational analysis is carried out to compare 5-residue natural interruptions of type IV collagen to Alport syndrome mutations. Interruption sequences contain more small and charged residues, and less stable triplets, whereas mutation sequences contain more imino acid and hydrophobic residues, and more stable triplets. These results suggest the local environment of a sequence break may determine whether it can be accommodated or lead to disease.

To assess whether natural interruptions have length-dependent properties, interruptions of 1 to 9 residues in length are introduced within triple helical peptides. All peptides in this set show conformational perturbations from the standard triple helix, with greater perturbations for longer interruptions. The peptides containing 8- and 9-residue interruptions form fibrous structures that may contain -sheet structure. The presence of amyloidogenic sequences within or between triple-helix domains may play a role in molecular association to normal tissue structures.

The effects of interruptions and mutations on folding are investigated using recombinant bacterial collagen. Type IV collagen interruption sequences of 4 and 15 residues in length significantly delay in vitro refolding, with the longer interruption causing a greater delay. It is possible that folding of type IV collagen is already slow due to the natural interruptions, so that an additional delay from a mutation pushes the folding rate over a tolerance threshold. Introduction of a Gly substitution mutation in a protein containing a 4-residue interruption causes an additional delay in refolding, which supports a folding problem in the pathogenesis of Alport syndrome.

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