On spectral relaxation in proteins

Photochemistry and Photobiology, Oct 2000 by Lakowicz, Joseph R

For completeness we note that the ability to observe negative a; values can be affected by the choice of solvents. In particular, somewhat more negative factors were observed for NATA in isobutanol (69,70). Observation of negative a; values depends on optimal conditions for dipolar relaxation to result in an adequate spectral shift on an appropriate timescale.

During the previous years the DAS of numerous proteins have been reported (88-96). DAS of STP and multi-trp proteins are shown in Figs. 19 and 20, respectively. Examination of the DAS reveals that the longer wavelength DAS are almost invariably associated with the longer decay times. This correlation suggests that the longer wavelength DAS are in fact the result of spectral relaxation. If the DAS were due to individual trp residues, or different conformations around a single trp, then based on the summary in Fig. 18 one expects that some long lifetime DAS will be at shorter wavelengths. The fact that such viability is not observed suggests the dominant role of spectral relaxation in determining the apparent DAS of proteins.

It is important to notice that DAS can be found for STP (Fig. 19). This is not expected for a single trp residue in a unique environment because NATA displays a single emission spectrum and decay time. It is also important to notice that the DAS of the STP are visually similar to the DAS found for NATA undergoing spectral relaxation (69). Figure 21 shows the DAS for NATA in propylene glycol at 0C. These DAS were calculated from the wavelength-dependent intensity decays (unpublished). In the absence of collisional quenchers the DAS show negative preexponential factors. Hence such factors can be expected under favorable conditions. Addition of collisional quenchers resuits in DAS that are all positive and resemble the DAS observed for proteins. This solution of NATA in a viscous solvent with nearby quenchers can be regarded as comparable to a trp residue in proteins with a nearby quenching group. These figures show how apparent DAS are easily observed even under conditions where discrete emitting species are not present. Given such results it is not realistic to conclude that the calculation of DAS proves that a protein exists in multiple conformations, and that these conformations always have longer emission maxima associated with the longer lifetime. The dominance of longer wavelength DAS with longer decay times for STP suggest that time-dependent spectral relaxation contributes significantly to the recovered DAS.

DAS have also been calculated for multi-trp proteins. Examples are shown in Fig. 20. In the case of the lac repressor the two DAS may be reasonably assigned to each of the two trp residues. In the case of phosphoglycerate kinase three DAS were recovered even though the protein contains only two trp residues. Once again one notices the usual trend that the DAS associated with longer wavelengths are those that display longer decay times. Since there does not appear to be any correlation between the trp emission maxima and decay times, there is no reason to expect the longer wavelength DAS to display longer lifetimes. These results suggest that spectral relaxation plays a role in the DAS recovered from both single and multi-trp proteins.