Characterization of the unfolding, refolding, and aggregation pathways of two protein implicated in cataractogenesis : human gamma D and human gamma S crystallin

Human [gamma]D crystallin (H[gamma]D-Crys) and human [gamma]S crystallin (H[gamma]S-Crys), are major proteins of the human eye lens and are components of cataracts. H[gamma]D-Crys is expressed early in life in the lens cortex while H[gamma]S-Crys is expressed throughout life in the lens epithelial cells. Both are primarily β-sheet proteins made up of four Greek keys separated into two domains and display 69% sequence similarity. The unfolding and refolding of H[gamma]D-Crys and H[gamma]S-Crys have been characterized as a function of guanidinium hydrochloride (GdnHCl) concentration at neutral pH and 37⁰C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Equilibrium unfolding and refolding experiments with GdnHCl showed unfolded protein is more fluorescent than its native counter-part despite the absence of metal or ion-tryptophan interactions in both of these proteins. This fluorescence quenching may influence the lens response to ultraviolet light radiation or the protection of the retina from ambient ultraviolet damage. Wild-type H[gamma]D-Crys exhibited reversible refolding above 1.0 M GdnHCl. Aggregation of refolding intermediates of H[gamma]D-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GdnHCl, beyond the major conformational transition region. H[gamma]S-Crys, however, exhibited a two-state reversible unfolding and refolding with no evidence of aggregation. Atomic force microscopy of H[gamma]D-Crys samples under aggregating conditions revealed ordered fiber structures that could recruit H[gamma]S-Crys to the aggregate. To provide fluorescence reporters

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Kosinski-Collins, Melissa Sue, 1978-

Advisor dc:contributor.advisor

• Jonathan King.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/28672
• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Identifier URI

http://dspace.mit.edu/handle/1721.1/28672

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/28672