On the involvement of electron transfer reactions in the fluorescence decay kinetics heterogeneity of proteins

Abstract

Time-resolved fluorescence study of single tryptophan-containing proteins, nuclease, ribonuclease T1, protein G, glucagon, and mastoparan, has been carried out. Three different methods were used for the analysis of fluorescence decays: the iterative reconvolution method, as reviewed and developed in our laboratory, the maximum entropy method, and the recent method that we called "energy transfer" method. All the proteins show heterogeneous fluorescence kinetics (multiexponential decay). The origin of this heterogeneity is interpreted in terms of current theories of electron transfer process, which treat the electron transfer process as a radiationless transition. The theoretical electron transfer rate was calculated assuming the peptide bond carbonyl as the acceptor site. The good agreement between experimental and theoretical electron-transfer rates leads us to suggest that the electron-transfer process is the principal quenching mechanism of Trp fluorescence in proteins, resulting in heterogeneous fluorescence kinetics. Furthermore, the origin of apparent homogeneous fluorescence kinetics (monoexponential decay) in some proteins also can be explained on the basis of electron-transfer mechanism.

Fig. 1.

Temperature dependence of the short lifetime recovered by maximum entropy method (solid square) and iterative reconvolution method (open square). The curves through the points are the least-squares fits analyzed as Ln(1/τ₁) vs 1/T.

Fig. 2.

Variation of theoretical ratio of k_ET (A) as function of dielectric constant of proteins models, N, Nucl; R, RNT1; P, ProtG; G, Gluc; and M, Mastp; where in the case of Gluc and Mastp, the ɛ was fixed to ɛ = 78. (B) as function of distance R between donor and acceptor. R_structure represents the shortest distance calculated from the protein structure.

Fig. 3.

Plot of the experimental (with maximum entropy method (MEM), iterative reconvolution method [IRM], and energy transfer method [ETM]) and theoretical ratio (electron transfer [ET] theory) of k_ET at T₁ = 10°C and T₂ = 40°C as function of the protein seize. N, Nucl; R, RNT1; P, ProtG; G, Gluc; and M, Mastp. Using for MEM and IRM (A) equation 4, and (B) equation 5. ETM and ET theory ratio are the same in both panels (A) and (B). The error calculations of the experimental ratio were done according to the incertitude propagation law (Taylor 1982).