On spectral relaxation in proteins

Photochemistry and Photobiology, Oct 2000 by Lakowicz, Joseph R

On Spectral Relaxation in Proteins^ para ||

ABSTRACT

During the past several years there has been debate about the origins of nonexponential intensity decays of intrinsic tryptophan (trp) fluorescence of proteins, especially for single tryptophan proteins (STP). In this review we summarize the data from diverse sources suggesting that time-dependent spectral relaxation is a ubiquitous feature of protein fluorescence. For most proteins, the observations from numerous laboratories have shown that for trp residues in proteins (1) the mean decay times increase with increasing observation wavelength; (2) decay associated spectra generally show longer decay times for the longer wavelength components; and (3) collisional quenching of proteins usually results in emission spectral shifts to shorter wavelengths. Additional evidence for spectral relaxation comes from the time-resolved emission spectra that usually shows time-dependent shifts to longer wavelengths. These overall observations are consistent with spectral relaxation in proteins occurring on a subnanosecond timescale. These results suggest that spectral relaxation is a significant if not dominant source of nonexponential decay in STP, and should be considered in any interpretation of nonexponential decay of intrinsic protein fluorescence.

INTRODUCTION

Numerous books, reviews and publications describe the intrinsic tryptophan (trp)$ fluorescence of proteins (1-6). While many specific molecular interactions that affect protein fluorescence are known, these factors are often considered individually as related to a particular protein. In the following sections we examine the evidence for time-dependent spectral shifts as the origin for multiexponential or nonexponential decays of trp fluorescence in proteins, with an emphasis on proteins containing a single trp residue. An overview of these data suggests that time-dependent spectral relaxation occurs in most proteins. The timescale of spectral relaxation appears to be somewhat faster than the mean decay times, but is slow enough to result in the multiexponential intensity decays typically found for single and multi-trp proteins. Subnanosecond spectral relaxation is consistent with extensive data from physical chemistry showing that spectral relaxation proceeds faster than the rotational diffusion of solvent molecules. While exceptions undoubtedly exist for completely buried or exposed residues, the more common partially exposed trp residues in most proteins probably display time-dependent shifts. The presence of time-dependent spectral relaxation should be considered in the interpretation of protein intensity decays and in the resolution of multiple trp residues in proteins.

TRYPTOPHAN AND PROTEIN FLUORESCENCE

Trp and the rotamer model

Interpretation of the intensity decays of proteins starts with an understanding of trp fluorescence. It is valuable to review what has been learned about the intensity decay of trp and its derivatives. It is well known that trp itself in neutral aqueous solution displays a double exponential intensity decay (7-12). A typical time-domain (TD) intensity decay is shown in Fig. 1 (13). Trp in aqueous solution is seen to be a weak double exponential, meaning a single decay time is a reasonable approximation of the data. It was difficult for the experimentalists to reach a consensus on the decay times and amplitudes because the short decay time component (T2 = 0.53 ns) makes only a minor contribution to the steady state intensity, about 8% (Table 1). Also, this decay time is present mostly on the blue side of the trp emission spectrum (Fig. 2). Note that in Figure 2 the amplitude of this component is multiplied 10-fold. Some experiments with longer wavelength observation did not detect this component, resulting in disagreement between different reports.

It is now generally accepted that the multiexponential decay of trp is due to the presence of rotational isomers called rotamers (Scheme 1). The presence of a double exponential decay is seen by the slightly elevated value of the goodness-of-fit parameter XR for the two-decay time fit as compared to the one-decay time fit. It appears that the positively charged amino group is positioned over the indole ring in one of the isomers, which quenches the indole by an electron transfer mechanism. Evidence for this mechanism includes the observation that the lifetime of trp increases several fold when the cn-amino group is deprotonated (14), and the single exponential decay of the neutral trp derivative is N-acetyl-t,-trypto-- phanamide (NATA) (Fig. 1). In NATA the amino and carboxyl groups are uncharged due to acetylation of the amine and amide formation of the carboxyl group. Without the quenching interaction due to the amino group NATA displays a single exponential decay, and the goodness-of-fit is not improved by including a second decay time in the analysis.

The continued analysis of the intensity decays of trp is a suitable discussion topic for the experts in trp fluorescence. However, it is time to put this issue to rest with more general discussions of protein fluorescence. The intensity decay of NATA is a single exponential (Fig. 1), an observation confirmed in many laboratories (8-10,1517). The amino acid residue in proteins is NATA, not trp, and the double exponential decay of trp is not relevant in consideration of multiexponential decays in proteins. For a single trp residue in a protein, in a single conformation with no time-dependent spectral relaxation, one expects a single exponential decay. Any deviations from a single exponential decay must have its origins in multiple conformations, protein dynamics, spectral relaxation, the presence of nearby quenchers or some other molecular interaction. Static quenching or a single resonance energy transfer (RET) acceptor at a single distance will result in a single rate constant depopulating the excited state, and the trp residue will still display a single exponential decay. Multiexponential decays can result from multiple ground state conformations, by dynamic motions of quenchers, acceptors or other residues surrounding the trp residue, or from timedependent spectral relaxation.