PMID- 18795792
OWN - NLM
STAT- MEDLINE
DCOM- 20081230
LR  - 20211020
IS  - 1520-4995 (Electronic)
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 47
IP  - 40
DP  - 2008 Oct 7
TI  - Mechanism of the efficient tryptophan fluorescence quenching in human 
      gammaD-crystallin studied by time-resolved fluorescence.
PG  - 10705-21
LID - 10.1021/bi800499k [doi]
AB  - Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens 
      protein found in the lens nucleus. Its long-term solubility and stability are 
      important to maintain lens transparency throughout life. HgammaD-Crys has four 
      highly conserved buried tryptophans (Trps), with two in each of the homologous 
      beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation 
      that reaches the lens. The dispersal of the excited-state energy to avoid 
      covalent damage is likely to be physiologically relevant for the lens 
      crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. 
      Previous steady-state fluorescence measurements 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 [Chen, J., et al. (2006) Biochemistry 45, 
      11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations 
      indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron 
      transfer to the amide backbone. Here we report additional information obtained 
      using time-resolved fluorescence spectroscopy. In the single-Trp-containing 
      proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly 
      quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, 
      whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau 
      approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the 
      lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to 
      approximately 1 ns. The intradomain energy transfer efficiency is 56% in the 
      N-terminal domain and is 71% in the C-terminal domain. The experimental values of 
      energy transfer efficiency are in good agreement with those calculated 
      theoretically. The absence of a time-dependent red shift in the time-resolved 
      emission spectra of Trp130 proves that its local environment is very rigid. 
      Time-resolved fluorescence anisotropy measurements with the single-Trp-containing 
      proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a 
      rigid body and no segmental motion is detected. A combination of energy transfer 
      with electron transfer results in short excited-state lifetimes of all Trps, 
      which, together with the high rigidity of the protein matrix around Trps, could 
      protect HgammaD-Crys from excited-state reactions causing permanent covalent 
      damage.
FAU - Chen, Jiejin
AU  - Chen J
AD  - Department of Biology, Massachusetts Institute of Technology, Cambridge, 
      Massachusetts 02139, USA.
FAU - Toptygin, Dmitri
AU  - Toptygin D
FAU - Brand, Ludwig
AU  - Brand L
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.
DEP - 20080917
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (gamma-Crystallins)
RN  - 8DUH1N11BX (Tryptophan)
SB  - IM
MH  - Humans
MH  - Protein Structure, Secondary
MH  - Spectrometry, Fluorescence/*methods
MH  - Tryptophan/*chemistry
MH  - gamma-Crystallins/*chemistry
PMC - PMC2758765
EDAT- 2008/09/18 09:00
MHDA- 2008/12/31 09:00
CRDT- 2008/09/18 09:00
PHST- 2008/09/18 09:00 [pubmed]
PHST- 2008/12/31 09:00 [medline]
PHST- 2008/09/18 09:00 [entrez]
AID - 10.1021/bi800499k [doi]
PST - ppublish
SO  - Biochemistry. 2008 Oct 7;47(40):10705-21. doi: 10.1021/bi800499k. Epub 2008 Sep 
      17.