John Domanick Labs 2-10 Report

John Domanick Labs 2-10 Report - The Effect of a V170R...

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The Effect of a V170R Mutation on HγD-Crystallin Conformation and Aggregate Formation of Cataracts By John Domanick, Gaspar Obimba, and Akash Vadalia
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Abstract Effect of V170R Mutation on HγD-crystallin stability J. Domanick, G. Obimba, A. Vadalia, Department of Biology, Brandeis University Cataracts formed from aggregates of HγD-crystallin have been shown to be caused by structural changes in the protein due to residue mutations that decrease the stability of the protein. We investigated the effect of the mutation V170R of the conformation of HγD-crystallin, and aggregate formation. Using site-directed mutagenesis to engineer a mutant form of the CRYGD gene, and bacterial transformation to produce the mutant HγD-crystallin we were able to use fluorescence to observed conformational changes due to our mutation. As the conformation of the mutant protein differs from the wild-type conformation the maximum fluorescence exhibited by the protein increases. The V170R mutant HγD-crystallin exhibits slightly elevated fluorescence values, showing small conformational changes. The V170R mutant does not drastically decrease HγD-crystallin stability nor promote cataract formation. Further studies will assess if the number of mutations can increase the odds of cataract formation.
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Introduction Human Gamma-D Crystallin (HγD-Crys) is a protein found in the lens of the eye, in relatively high concentrations. HγD-Crys serves to refract incoming light and focus it onto the retina to grant vision to the eye. This function is known to arise from the shape and structure of HγD-Crys, and the maintenance of this structure is crucial to maintaining vision. Protein aggregation is the consequence of the incorrect folding of proteins in a cell, which can be caused by many factors, including the alteration of protein primary structures due to a mutation in DNA (Kopito 2000). If the structure of HγD-Crys becomes sufficiently disrupted it will aggregate in the eye and form a cataract, thus degrading vision in that eye. Several mutations found in the HγD- Crys coding gene, CRYGD, have been identified with causing cataracts. This experiment served to investigate the mutation V170R, wild-type valine at position 170 changed to arginine, and its affect on the structure of HγD-Crys, and if it was a possible cataract forming mutation. HγD-Crys is a protein with two domains, with a central interaction of hydrophobic residues, methionine, phenylalanine, isoleucine, leucine, and valine, in between the two domains. It was hypothesized that a mutation of a residue involved in this interaction would severely disrupt the structural integrity of HγD-Crys. It was thought that substituting the large, basic residue, arginine, for the small, hydrophobic residue valine would repulse the other hydrophobic residues enough to break open the HγD-Crys, analogous to the rupturing of a popcorn kernel. Such a large
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This note was uploaded on 10/06/2011 for the course BIOL 18 taught by Professor Kc during the Fall '10 term at Brandeis.

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John Domanick Labs 2-10 Report - The Effect of a V170R...

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