On spectral relaxation in proteins

Photochemistry and Photobiology, Oct 2000 by Lakowicz, Joseph R

The application of these concepts to protein spectral relaxation is shown in Fig. 26. For trp red-edge shifts are only seen below 20C in glycerol (left). Hence, one can use the red-edge shift as a measure of the dynamics of the environment surrounding trp residues. Red-edge shifts for a number of STP are also shown in Fig. 26 (right). If the trp residue is in a completely nonpolar environment, as for whiting parvalbumin and ribonuclease T,, there is no red-edge shift and no information on dynamics. For most of the other proteins there is a significant red-edge shift, the most dramatic being seen for human serum albumin. As the residues become completely exposed to the solvent, the red-edge shifts disappear, as for the melittin monomer. In this case the local dynamics are faster than the mean decay time. An overall conclusion from Fig. 26 is that spectral relaxation is comparable to the decay time in about half of the proteins. Once again this result indicates that spectral relaxation should be considered in any analysis of protein DAS.

DISCUSSION

DAS of genetically engineered proteins

The preceding sections were strongly worded to emphasize the important role of spectral relaxation in proteins, and to indicate the possibility of overinterpretation of the DAS when spectral relaxation is ignored. However, it is important to acknowledge and recognize the number of elegant studies in which the decay components have reliably been assigned to individual trp residues (123-125). Such results are typically obtained for two or three trp proteins in which the individual residues have been removed by site-directed mutagenesis, followed by expression of the single tip mutant proteins. In such cases one can determine the spectral properties of residues and occasionally observe interactions between the residues and/or neighboring groups by RET or quenching.

Site-directed mutagenesis has provided important insights into the relationship of protein structure and the spectral properties of the trp residues. These studies have provided definitive evidence of quenching by nearby protonated imidazole, carboxyl and amino groups, phenyl groups and disulfide bonds (108-111). It is valuable to use these known interactions for understanding the structure and dynamics of individual proteins. In fact, these insights have increased the power and information content of the fluorescent data. The presently available information on the effects of environment on trp fluorescence, and the susceptibility of trp to quenching interactions, allows reliable use of protein fluorescence to learn about proteins.

Protein fluorescence is not particle physics

In some fields of science, observation of a single exception to the theory results in invalidation of the theory and to a search for improved models. This principle does not apply to protein fluorescence. There is little doubt that exceptions to the general thesis of this article will be found, i.e. longer lifetimes at shorter wavelengths or longer lifetime DAS at shorter wavelengths. In fact, there are a few reports on shorter wavelength DAS for longer decay times (126,127). Observations of such exceptional proteins will remain just that, exceptions. The evidence from a large number of proteins is strong evidence for the important role of spectral relaxation in proteins. Interpretation of the time-resolved data should start with consideration that the observed properties could be the result of spectral relaxation. Failure to do so can result in the creation of models, typically containing discrete conformations, which have not been demonstrated by the data. Acknowledgements-This work was supported by the NIH, National Center for Research Resources, RR-08119. The author also acknowledges prior support from the NIH and the NSF, under which many of these concepts were developed. Special thanks is given to Dr. Kazik Nowaczyk and Dr. Ignacy Gryczynski for calculation of the DAS and emission centers-of-gravity.