TY - JOUR
T1 - Tryptophan dynamics of the FK506 binding protein
T2 - Time-resolved fluorescence and simulations
AU - Silva, Norberto D.
AU - Prendergast, Franklyn G.
N1 - Funding Information:
This work was supported in part by the Mayo Foundation, by grant GM 34847 (to FGP) and by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG-1289 (to NS). Thanks are expressed to Dr. A. Marcy and Dr. B. McKeever of Merck Research Laboratories for providing the human FKBP12 plasmid and the crystal coordinates of uncomplexed human FKBP12. Thanks also to Dr. T. Felmee, Dr. D. Braddock, Dr. C. Haydock, Dr. Y-P. Pang, Ms. S. Kurian, Dr. W. Kirk, and Mr. T. Wieand for their technical advice.
PY - 1996/3
Y1 - 1996/3
N2 - The FK506-binding protein (FKBP12) is important in the immunosuppressant action of FK506 and rapamycin. We have investigated Trp side chain dynamics in FKBP12, with and without a bound immunosuppressant, by measuring the Trp time-resolved fluorescence anisotropy decay r(t). The r(t) for W59 in aqueous uncomplexed FKBP12 at 20°C is well described by a single exponential with a recovered initial anisotropy, r(o)/(eff), of 0.192 and an overall rotational correlation time for the protein, φ(p), of 4.7 ns; r(o)/(eff) = 0.214 and φ(p) = 4.2 ns for the FKBP12/FK506 complex. Using an expression for the order parameter squared, namely S2 = r(o)/(eff)/r(o)/(T), where r(o)/(T) is the vitrified steady-state excitation anisotropy, we recovered an S2 of 0.75 for W59 fluorescence in uncomplexed FKBP12 and S2 ≃ 1 in the FKBP12/FK506 complex. Results obtained for the FKBP12/rapamycin complex are similar to those found for the FKBP12/FK506 complex. Minimum perturbation mapping simulations were performed on the free and complexed forms of FKBP12 and the results were generally in agreement with the experimental data.
AB - The FK506-binding protein (FKBP12) is important in the immunosuppressant action of FK506 and rapamycin. We have investigated Trp side chain dynamics in FKBP12, with and without a bound immunosuppressant, by measuring the Trp time-resolved fluorescence anisotropy decay r(t). The r(t) for W59 in aqueous uncomplexed FKBP12 at 20°C is well described by a single exponential with a recovered initial anisotropy, r(o)/(eff), of 0.192 and an overall rotational correlation time for the protein, φ(p), of 4.7 ns; r(o)/(eff) = 0.214 and φ(p) = 4.2 ns for the FKBP12/FK506 complex. Using an expression for the order parameter squared, namely S2 = r(o)/(eff)/r(o)/(T), where r(o)/(T) is the vitrified steady-state excitation anisotropy, we recovered an S2 of 0.75 for W59 fluorescence in uncomplexed FKBP12 and S2 ≃ 1 in the FKBP12/FK506 complex. Results obtained for the FKBP12/rapamycin complex are similar to those found for the FKBP12/FK506 complex. Minimum perturbation mapping simulations were performed on the free and complexed forms of FKBP12 and the results were generally in agreement with the experimental data.
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U2 - 10.1016/S0006-3495(96)79706-0
DO - 10.1016/S0006-3495(96)79706-0
M3 - Article
C2 - 8785272
AN - SCOPUS:0030052607
SN - 0006-3495
VL - 70
SP - 1122
EP - 1137
JO - Biophysical Journal
JF - Biophysical Journal
IS - 3
ER -