Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.
We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.
Ross
J B
JB
Department of Biochemistry, Mount Sinai School of Medicine, New York, NY 10029.
Senear
D F
DF
Waxman
E
E
Kombo
B B
BB
Rusinova
E
E
Huang
Y T
YT
Laws
W R
WR
Hasselbacher
C A
CA
D006916:5-Hydroxytryptophan / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004735:Energy Transfer; D005454:Fluorescence Polarization; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012997:Solvents; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D014443:Tyrosine; D006916:5-Hydroxytryptophan