Mechanism of the efficient quenching of tryptophan fluorescence in human gamma crystallin

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human [gamma]D-crystallin (H[gamma]D-Crys) and human [gamma]S-crystallin (H[gamma]S-Crys) are both very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged H[gamma]D-Crys or H[gamma]S-Crys is associated with the prevalent eye disease mature-onset cataract. Both H[gamma]D-Crys and H[gamma]S-Crys have two homologous [beta]-sheet domains, each containing a pair of highly conserved buried tryptophans (see Fig. 1). The overall fluorescence of the Trps is quenched in the native state of H[gamma]D-Crys N-terminal domain C-terminal domain and H[gamma]S-Crys. In crystallin proteins, these Trps will Tr56 be absorbing UV radiation that reaches the lens. The dispersal of the excited state energy is likely to be H[gamma]siological relevant for the lens crystallins. Trp42 Steady-state and time-resolved fluorescence measurements combined with H[gamma]brid quantum Figure 1: The crystal structure of wild- mechanical-molecular mechanical (QM-MM) type H[gamma]D-Crys depicted in ribbon simulations revealed the quenching mechanism of representation showing the four H[gamma]D-Crys. From fluorescence of triple Trp to Phe intrinsic tryptophans in spacefill, mutants, the homologous pair Trp68 and Trpl56 are Trp42 and Trp68 in the N-terminal domain and Trpl30 and Trp156 in the found to be extremely quenched, with quantum C-terminal domain (Protein Data Bank yields close to 0.01, and with very short lifetimes, Code: 1HKO). T-0. 1ns. In contrast, the homologous pair Trp42 and Trpl30 are moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively, and with longer lifetimes, T-3ns. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or H[gamma]drophobic residues.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Chen, Jiejin, Ph. D. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• Jonathan King.

• Chemistry.

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/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/45428

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/45428