Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein.
The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins.
Hasselbacher
C A
CA
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Rusinova
E
E
Waxman
E
E
Rusinova
R
R
Kohanski
R A
RA
Lam
W
W
Guha
A
A
Du
J
J
Lin
T C
TC
Polikarpov
I
I
D001483:Base Sequence; D002942:Circular Dichroism; D004355:Drug Stability; D015942:Factor VIIa / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence; D013925:Thromboplastin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014364:Tryptophan; D014443:Tyrosine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D013925:Thromboplastin; D015942:Factor VIIa