PHOSPHORESCENCE AND OPTICALLY DETECTED MAGNETIC-RESONANCE CHARACTERIZATION OF THE ENVIRONMENTS OF TRYPTOPHAN ANALOGS IN STAPHYLOCOCCAL NUCLEASE, ITS V66W MUTANT, AND DELTA-137-149 FRAGMENT

Phosphorescence and optically detected magnetic resonance (ODMR) measu rements are reported on the triplet states of the tryptophan analogues , 7-azatryptophan (7AW), 5-hydroxytryptophan (5HW), and 4-, 5-, and 6- fluorotryptophan (4FW, 5FW, 6FW) when incorporated at position 140 of wild-type Staphylococcal nuclease (7AW-nuclease, etc.), positions 66 a nd 140 of its V66W mutant (7AW-V66W, etc.), and the deletion fragment of the latter, Delta 137-149 (7AW-V66W', etc.). These measurements poi nt to the retention of protein structure at position 140 in each of th e wild-type nuclease analogues. Substitution of the analogue at both t ryptophan sites of V66W leads to structured sites with differentiated triplet-state properties for all analogues except 7AW-V66W, whose stru cture is destabilized, 5HW-V66W' is the only fragment that apparently lacks structure at position 66. All other V66W' analogues exhibit a st ructured environment at position 66 (4FW-V66W' was not studied), but i n each case this site can be differentiated readily from the correspon ding site in intact V66W. 7AW-V66W' is resolved by ODMR into two discr ete structures with slightly differing zero field splittings (ZFS). In teraction of the protein with 5HW at position 66 of 5HW-V66W induces a 2-fold increase in the ZFS E parameter, which is reduced to its norma l value upon formation of the fragment, 5HW-V66W'. Analogous effects o ccur for 5FW, but on a smaller scale.