Senior Honors Projects, 2010-current

Preferred Name - First Author

Michael Rudloff

Date of Award

Spring 2015

Document Type

Dissertation/Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Biology

Advisor(s)

Nathan Wright

Jonathan Monroe

Abstract

The giant human muscle proteins titin and obscurin are important for sarcomeric organization, stretch response, and sarcomerogenesis in myofibrils. The extreme C-terminus of titin (the M10 domain) binds to the N-terminus of obscurin (the Ig1 domain) in the M-line, an interaction that is critical for sarcomere stability. The high-resolution structure of human M10 has been solved, along with M10 bound to one of its two known molecular targets, the Ig1 domain of obscurin-like protein. Multiple M10 mutations are linked to limb-girdle muscular dystrophy type 2J (LGMD2J) and tibial muscular dystrophy (TMD), however the effect of the M10 mutations on protein structure and function has not been thoroughly characterized. Here, all four naturally occurring human M10 missense mutants have been engineered and biophysically characterized in vitro with the hypothesis being the mutations ablate this critical interaction. Two of the four mutated constructs are severely misfolded and cannot bind to the obscurin Ig1 domain. One mutation, H66P, is folded at room temperature but unfolds at 37 oC, rendering it binding incompetent. The I57N mutation shows no significant structural, dynamic, or binding differences from the wild-type domain. We suggest that this mutation is not directly responsible for muscle wasting disease, but is instead merely a polymorphism found in symptomatic patients. Understanding the biophysical basis of muscle wasting disease can help streamline potential future treatments.

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