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

Abstract

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 ( approximately 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 (R(0)) was determined, by using the known x-ray data, to be 19.5 A for Trp-36 and 25.2 A 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.

Figure 1

Ribbon presentation of high-resolution x-ray structure of hyperthermophilic Pyrococcus furiosus rubredoxin (11). Six aromatic amino acid residues in the hydrophobic core are, along the primary sequence, Trp-3, Tyr-10, Tyr-12, Phe-29, Trp-36, and Phe-48. A tail at the C terminus protrudes into the solvent.

Figure 2

Absorption of the PfRd protein and normalized fluorescence emission for different systems. Note that the spectral overlap between the Trp emission in apo-PfRd and the [Fe-S] cluster absorption in PfRd. The fluorescence intensity in PfRd is actually much weaker than that in apo-PfRd. The arrows mark two excitation wavelengths used in this study, 265 nm and 288 nm.

Figure 3

(A) Normalized, femtosecond-resolved fluorescence decay of Trp in buffer solution at pH 2 with a series of wavelength detection. (Inset) The constructed solvent response c(t). Normalized fluorescence decay of Trp in the apo-fmetPfRd protein (B) and in its denatured state (C). Note that the transient at 340-nm emission in B decays only with a long lifetime of ≈530 ps.

Figure 4

Normalized, femtosecond-resolved fluorescence decay of Trp in the fmetPfRd protein at 288-nm excitation with a series of wavelength detection at long time scale (A) and for the initial part (B). Note that at wavelengths longer than 340 nm, a constant signal was observed up to 3 ps.

Figure 5

(A) Normalized, femtosecond-resolved fluorescence transients of Trp in various systems and under different conditions for 340-nm detection at 265-nm excitation. The corresponding initial parts are shown in B. Note that only the wild-type PfRd and the variant fmetPfRd at pH 7 have an initial constant signal for 2–3 ps; see text for detail.

Figure 6

(A) Femtosecond-resolved fluorescence transients of Trp in water at parallel and perpendicular conditions (Upper) for 340-nm emission at 265-nm excitation and the corresponding anisotropy decay (Lower). (Inset) The anisotropy decay for the long time scale. (B) Femtosecond-resolved fluorescence transients of Trps in the fmetPfRd protein at parallel and perpendicular conditions (Upper) for 310-nm emission at 288-nm excitation and the corresponding anisotropy decay. The anisotropy at 340-nm emission shows an identical temporal behavior (not shown). Note that the anisotropy of Trps in the protein stays constant on the picosecond time scale.