Analysis of internal motion of single tryptophan in Streptomyces subtilisin inhibitor from its picosecond time-resolved fluorescence.
A mode of internal motion of single tryptophan, Trp 86, of Streptomyces subtilisin inhibitor, was analyzed from its time-resolved fluorescence. The intensity and anisotropy decays of Trp 86 were measured in the picosecond range. These decays were analyzed with theoretical expressions derived assuming that the indole ring of tryptophan as an asymmetric rotor rotates around covalent bonds connecting indole with the peptide chain and an effective quencher of fluorescence of Trp 86 is the nearby SS bond of Cys 35-Cys 50. First, the intensity decays at 6 degrees, 20 degrees, and 40 degrees C were analyzed, and then the both decays of the intensity and anisotropy at 20 degrees C were simultaneously simulated with common parameters. Constants concerning geometrical structures of the protein used for the analysis were obtained from x-ray crystallographic data. Best fit between the observed and calculated decay curves was obtained by a nonlinear least squares method by adjusting a quenching constant averaged over the rotational angles, koq height of the potential energy, p, and three of six diffusion coefficients, Dxx, Dyy, Dzz, Dxy, Dyz, and Dzx, as variable parameters. The obtained results revealed that the internal motion of the indole ring became faster, the quenching rate of the fluorescence of Trp 86 was enhanced and the height of potential energy became lower at higher temperatures, and suggested that Trp 86 was wobbling around the long axis of the indole ring in the protein.
Tanaka
F
F
Mie Nursing College, Tsu, Japan.
Tamai
N
N
Mataga
N
N
Tonomura
B
B
Hiromi
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D005454:Fluorescence Polarization; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013302:Streptomyces; D014364:Tryptophan
D001426:Bacterial Proteins; D015842:Serine Proteinase Inhibitors; C018150:subtilisin inhibitor protein, Streptomyces; D014364:Tryptophan