INCORPORATION OF TRYPTOPHAN ANALOGS INTO STAPHYLOCOCCAL NUCLEASE, ITSV66W MUTANT, AND DELTA-137-149 FRAGMENT - SPECTROSCOPIC STUDIES

We have biosynthetically incorporated several tryptophan analogues int o three forms of Staphylococcal nuclease to investigate the spectrosco pic characteristics of these ''intrinsic'' probes and their effect on the structure of the proteins. The set of tryptophan analogues include s 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotr yptophan, and 6-fluorotryptophan, 5-Hydroxytryptophan and 7-azatryptop han have red-shifted absorbance spectra, and the latter has a red-shif ted fluorescence, which is very sensitive to its environment (being he avily quenched in water). The fluorotryptophans can serve as F-19 NMR probes, and 4-fluorotryptophan has a very low fluorescence quantum yie ld, thus making it a ''knock-out'' fluorescence analogue. The set of p roteins studied includes wild-type nuclease, which has a single trypto phan site at position 140; its V66W mutant, which has a second tryptop han at position 66; and the Delta 137-149 fragment, V66W', which only has a tryptophan at position 66. The environments of positions 66 and 140 are significantly different; position 140 is near the end of the l ong C-terminal alpha-helix and is moderately solvent-exposed, whereas position 66 is in the beta-barrel core region of the protein and is su rrounded by apolar side chains. Absorbance and F-19 NMR spectra are us ed to estimate the extent of analogue incorporation for each protein. Steady-state and time-resolved fluorescence data are reported to chara cterize the emission of the analogues in these positions in the three proteins and to develop the use of the analogues as probes of protein structure and dynamics. Circular dichroism spectra are reported to sho w that, in all but a couple of cases, the secondary structure of the p roteins containing the analogues is not significantly perturbed by the probes. Additionally, fluorescence anisotropy decay data show the var iants of wild-type nuclease to have a rotational correlation time simi lar to that of tryptophan-containing nuclease.