Fluorescence properties of recombinant tropomyosin containing tryptophan, 5-hydroxytryptophan and 7-azatryptophan.
Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptophan (5OH122W) or 7-azatryptophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent-exposed position c of the coiled-coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue-shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2-3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple-exponential fluorescence decay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7-azatryptophan. The range of lifetimes observed for 5-hydroxyindole and 5-hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 5OH122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 5OH122W.
Das
K
K
Department of Chemistry, Iowa State University, Ames 50011-3111, USA.
Ashby
K D
KD
Smirnov
A V
AV
Reinach
F C
FC
Petrich
J W
JW
Farah
C S
CS
D006916:5-Hydroxytryptophan / Q000737:chemistry; D000818:Animals; D002645:Chickens; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D014335:Tropomyosin; C006657:7-azatryptophan; D014364:Tryptophan; D006916:5-Hydroxytryptophan