DISTANCE DISTRIBUTIONS FROM THE TYROSYL TO DISULFIDE RESIDUES IN THE OXYTOCIN AND [ARG(8)]-VASOPRESSIN MEASURED USING FREQUENCY-DOMAIN FLUORESCENCE RESONANCE ENERGY-TRANSFER

We have examined the fluorescence intensity decays of oxytocin and [Ar g(8)]-vasopressin resulting from the single tyrosyl residue in each pe ptide, and the intensity decay of the Asu(1,6)-analogues in which the disulfide bridge is substituted by a CH2-CH2 bridge. Viscosity-depende nt steady state and intensity decay measurements indicated that fluore scence resonance energy transfer (FRET) from tyrosyl phenol to the dis ulfide bridge is responsible for the decrease in fluorescence relative to the Asu-analogues. The frequency-domain phase and modulation data for the tyrosyl donor were interpreted in terms of fluorescence resona nce energy transfer (FRET) to the weakly absorbing disulfide bridge an d a distribution of donor-to-acceptor distances. Energy transfer effic iencies were determined from both time-resolved and steady-state measu rements. Fitting the frequency-domain phase and modulation data to a G aussian distance distribution indicated that the average inter-chromop horic distance (R(av)) is similar in both compounds, R(av)=7.94 Angstr om for oxytocin and R(av) = 8.00 Angstrom for vasopressin. However, th e width of the distance distribution is narrower for vasopression (hw= 2.80 Angstrom) than for oxytocin (hw=3.58 A), which is consistent with restriction of the tyrosine phenol motion due to its stacking with th e Phe(3) side chain of vasopressin. Finally, the recovered distance di stribution functions are compared with histograms describing the dista nce between the chromophores during the course of long, in vacuo, mole cular dynamics runs using the computer program CHARMm and the QUANTA 3 .0 parameters.