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 (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.
Ozarowski
A
A
Department of Chemistry, University of California, Davis 95616, USA.
Wu
J Q
JQ
Davis
S K
SK
Wong
C Y
CY
Eftink
M R
MR
Maki
A H
AH
D006916:5-Hydroxytryptophan / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics*; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics; D014633:Valine / Q000235:genetics*
D010446:Peptide Fragments; C006657:7-azatryptophan; C039390:4-fluorotryptophan; C026927:5-fluorotryptophan; C001244:6-fluorotryptophan; D014364:Tryptophan; D006916:5-Hydroxytryptophan; D008836:Micrococcal Nuclease; D014633:Valine