Title:
Steps to Improving Stability of the β-propeller Structure of Myocilin's Olfactomedin Domain: Understanding the Evolution of the β-propeller
Steps to Improving Stability of the β-propeller Structure of Myocilin's Olfactomedin Domain: Understanding the Evolution of the β-propeller
Author(s)
Kwon, Michelle S.
Advisor(s)
Lieberman, Raquel L.
Williams, Loren D.
Williams, Loren D.
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Abstract
Olfactomedin (OLF) domain-containing proteins, first identified in relation to bullfrog olfactory chemoreception, are part of a superfamily of proteins implicated in many important biological functions and human diseases. The myocilin OLF domain (mOLF), one of the best studied, is closely associated with the ocular disease glaucoma. Nearly 100 myocilin mutations have been reported in glaucoma patients; >90% are missense mutations within mOLF. Disease-associated mutant myocilins are destabilized and aggregation prone, leading to toxicity and death of cells that maintain the anatomical trabecular meshwork extracellular matrix in the eye. The Lieberman lab solved the crystal structures of OLF domains from myocilin and gliomedin (gOLF), a peripheral nervous system OLF domain. While both are similar five-bladed β-propellers, only mOLF contains a stabilizing calcium ion. Remarkably, gOLF is ~20 °C more stable than mOLF, even though it doesn't have a calcium ion and is phylogenetically more primitive. The goal of this project was to use insights from mOLF and gOLF to create a thermostable mOLF. Surprisingly, mutagenesis of a calcium-coordinating aspartate (D478) to alanine abolished calcium binding but increased mOLF thermal stability to near gOLF levels. Addition of D478A to the destabilized, glaucoma-associated variant D380A rescued thermal stability to that of wild-type (WT) mOLF. Structures of thermostable mOLF variants reveal unexpected changes in tertiary structure compared to WT mOLF, which were confirmed by solution biophysical measurements. The findings from this study expand our understanding of the structure-stability relationship of mOLF and provide further insight into the evolution of the OLF β-propeller.
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Date Issued
2017-05
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Text
Resource Subtype
Undergraduate Thesis