(Georgia Institute of Technology, 2017-05)
Kwon, Michelle S.
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.
(Georgia Institute of Technology, 2016-01-25)
Heaner, David Prince
The determination of membrane protein structures is critical for the development of new pharmaceutical agents. Conventionally, membrane proteins are solubilized by the use of mild detergents. However, due to the lack of hydrophilic residues available to make crystal contacts and interference by the large detergent micelle, the quality of diffraction and resolution level needed for de novo structure determination is usually not obtained. In addition to the conventional detergent method, a new method using engineered single chain antibody fragments (scFv) and a Fab antibody fragment have been developed for use as crystallization chaperones. The scFv and Fab fragment interact with the membrane protein of interest via the EYMPME (EE) tag, which is selectively mutated into a hydrophilic loop of the protein. The membrane protein-antibody fragment complex may enter crystallization trials with the antibody fragment driving the complex nucleation through the formation of numerous, strong crystal contacts. Such a co-crystallization method with anti-EE scFv and Fab fragments provides the protein crystallographer with a “crystallization toolbox” that can be used for any crystallographic study of a protein of interest. Through size exclusion chromatography and SDS-PAGE analysis, complexation of the β-barrel membrane protein intimin harboring the EE tag with scFv/EE and Fab/EE has been shown to occur, from which crystallization trials have ensued. Formation of a co-crystal has proven to be difficult, which can be explained in part through molecular dynamics simulations of the mutated intimin L4 loop. This thesis work will present results and conclusions for this novel co-crystallization method.