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Published January 8, 2002 | Published
Journal Article Open

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein


We report here studies of tryptophan (Trp) solvation dynamics in water and in the Pyrococcus furiosus rubredoxin protein, including the native and its apo and denatured forms. We also report results on energy transfer from Trp to the iron-sulfur [Fe-S] cluster. Trp fluorescence decay with the onset of solvation dynamics of the chromophore in water was observed with femtosecond resolution (approx 160 fs; 65% component), but the emission extended to the picosecond range (1.1 ps; 35% component). In contrast, the decay is much slower in the native rubredoxin; the Trp fluorescence decay extends to 10 ps and longer, reflecting the local rigidity imposed by residues and by the surface water layer. The dynamics of resonance energy transfer from the two Trps to the [Fe-S] cluster in the protein was observed to follow a temporal behavior characterized by a single exponential (15-20 ps) decay. This unusual observation in a protein indicates that the resonance transfer is to an acceptor of a well-defined orientation and separation. From studies of the mutant protein, we show that the two Trp residues have similar energy-transfer rates. The critical distance for transfer (R0) was determined, by using the known x-ray data, to be 19.5 Å for Trp-36 and 25.2 Å for Trp-3, respectively. The orientation factor (kappa 2) was deduced to be 0.13 for Trp-36, clearly indicating that molecular orientation of chromophores in the protein cannot be isotropic with kappa 2 value of 2/3. These studies of solvation and energy-transfer dynamics, and of the rotational anisotropy, of the wild-type protein, the (W3Y, I23V, L32I) mutant, and the fmetPfRd variant at various pH values reveal a dynamically rigid protein structure, which is probably related to the hyperthermophilicity of the protein.

Additional Information

© 2002, The National Academy of Sciences. Contributed by Ahmed H. Zewail, October 31, 2001. We like to thank Prof. Michael W. W. Adams and Dr. Francis E. Jenney, Jr. (University of Georgia) for the generous gift of all proteins reported here. We acknowledge the assistance of Dr. Spencer Baskin for the measuring of the lifetime of the apoprotein and for helpful discussion. This work was supported by the National Science Foundation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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