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  • Phosphorescence and optically detected magnetic resonance characterization of the environments of tryptophan analogues in staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment.

Phosphorescence and optically detected magnetic resonance characterization of the environments of tryptophan analogues in staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment.

Biochemistry (1998-06-24)
A Ozarowski, J Q Wu, S K Davis, C Y Wong, M R Eftink, A H Maki
ABSTRACT

Phosphorescence and optically detected magnetic resonance (ODMR) measurements 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 and 140 of its V66W mutant (7AW-V66W, etc.), and the deletion fragment of the latter, Delta 137-149 (7AW-V66W', etc.). These measurements point to the retention of protein structure at position 140 in each of the wild-type nuclease analogues. Substitution of the analogue at both tryptophan sites of V66W leads to structured sites with differentiated triplet-state properties for all analogues except 7AW-V66W, whose structure is destabilized. 5HW-V66W' is the only fragment that apparently lacks structure at position 66. All other V66W' analogues exhibit a structured environment at position 66 (4FW-V66W' was not studied), but in each case this site can be differentiated readily from the corresponding site in intact V66W. 7AW-V66W' is resolved by ODMR into two discrete structures with slightly differing zero field splittings (ZFS). Interaction 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 normal value upon formation of the fragment, 5HW-V66W'. Analogous effects occur for 5FW, but on a smaller scale.

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5-Fluoro-L-tryptophan, ≥98.0% (HPLC)
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Sigma-Aldrich
5-Fluoro-DL-tryptophan, powder or crystals
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