Date of Award

1-1-2011

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Chemistry

Content Description

1 online resource (ix, 56 pages) : illustrations (some color)

Dissertation/Thesis Chair

Jayanti Pande

Committee Members

Carla Theimer

Keywords

cataract, crystallin, mutation, protein, Cataract, Crystalline lens, Cataract in children

Subject Categories

Biochemistry | Chemistry

Abstract

Cataract disease results when the eye lens becomes opaque and scatters a significant part of the incoming light into the eye. The lens contains very high concentrations of the lens proteins, called crystallins, which are present at concentrations comparable to those found in protein crystals (about 400-600 mg/mL). Chemical modifications of the crystallins, such as oxidation and deamidation, or genetic mutations are known to result in increased light-scattering in vitro, and are implicated in cataract formation in vivo. Here we present the in vitro work on a mutant protein of human gammaD crystallin (HGD), namely R76S (i.e. Arg 76 to Ser substitution) which is associated with juvenile cataract. Our spectroscopic studies show that the mutant protein has secondary and tertiary structures identical to those of the native protein. Its thermal stability also appears to be unaltered by the mutation. The observed differences between the wild-type and mutant proteins are: (a) the pI of R76S is lower by 0.4 pH units relative to HGD, which is expected since the basic Arg residue is replaced by a neutral Ser, (b) small chemical shift differences in the HSQC spectra of the two proteins; which indicate that there are minor differences in their structure that are localized near the mutation site, and (c) the R76S mutant shows a slightly higher level of Ca2+ ion binding than the wild type, although the difference is less than 10%, and is unlikely to lead to lens opacity.

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