PMID- 16981715
OWN - NLM
STAT- MEDLINE
DCOM- 20061108
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 45
IP  - 38
DP  - 2006 Sep 26
TI  - Mechanism of the highly efficient quenching of tryptophan fluorescence in human 
      gammaD-crystallin.
PG  - 11552-63
AB  - Quenching of the fluorescence of buried tryptophans (Trps) is an important 
      reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a 
      very stable eye lens protein that must remain soluble and folded throughout the 
      human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is 
      associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has 
      two homologous beta-sheet domains, each containing a pair of highly conserved 
      buried tryptophans. The overall fluorescence of the Trps is quenched in the 
      native state despite the absence of the metal ligands or cofactors. We report the 
      results of detailed quantitative measurements of the fluorescence emission 
      spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. 
      From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and 
      Trp156 were found to be extremely quenched, with quantum yields close to 0.01. 
      The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum 
      yields of 0.13 and 0.17, respectively. 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 hydrophobic residues. None of 
      these mutants showed significant changes in the fluorescence intensity compared 
      to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) 
      simulations with the four different excited Trps as electron donors strongly 
      indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 
      are extremely fast relative to those for Trp42 and Trp130. This is in agreement 
      with the quantum yields measured experimentally and consistent with the absence 
      of a quenching side chain. Efficient electron transfer to the backbone is 
      possible for Trp68 and Trp156 because of the net favorable location of several 
      charged residues and the orientation of nearby waters, which collectively 
      stabilize electron transfer electrostatically. The fluorescence emission spectra 
      of single and double Trp to Phe mutants provide strong evidence for energy 
      transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 
      in the C-terminal domain. The backbone conformation of tryptophans in 
      HgammaD-Crys may have evolved in part to enable the lens to become a very 
      effective UV filter, while the efficient quenching provides an in situ mechanism 
      to protect the tryptophans of the crystallins from photochemical degradation.
FAU - Chen, Jiejin
AU  - Chen J
AD  - Department of Biology, Massachusetts Institute of Technology, Cambridge, 
      Massachusetts 02139, USA.
FAU - Flaugh, Shannon L
AU  - Flaugh SL
FAU - Callis, Patrik R
AU  - Callis PR
FAU - King, Jonathan
AU  - King J
LA  - eng
GR  - EY 015834/EY/NEI NIH HHS/United States
GR  - GM 17980/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (CRYGD protein, human)
RN  - 0 (Crystallins)
RN  - 0 (Mutant Proteins)
RN  - 0 (gamma-Crystallins)
RN  - 8DUH1N11BX (Tryptophan)
SB  - IM
MH  - Amino Acid Sequence
MH  - Amino Acid Substitution
MH  - Crystallins/chemistry/*metabolism
MH  - *Fluorescence
MH  - Fluorescence Resonance Energy Transfer
MH  - Humans
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutant Proteins/metabolism
MH  - Mutation/genetics
MH  - Protein Structure, Secondary
MH  - Protein Structure, Tertiary
MH  - Quantum Theory
MH  - Tryptophan/*metabolism
MH  - gamma-Crystallins
EDAT- 2006/09/20 09:00
MHDA- 2006/11/10 09:00
CRDT- 2006/09/20 09:00
PHST- 2006/09/20 09:00 [pubmed]
PHST- 2006/11/10 09:00 [medline]
PHST- 2006/09/20 09:00 [entrez]
AID - 10.1021/bi060988v [doi]
PST - ppublish
SO  - Biochemistry. 2006 Sep 26;45(38):11552-63. doi: 10.1021/bi060988v.