Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer.
We have examined the fluorescence intensity decays of oxytocin and [Arg8]-vasopressin resulting from the single tyrosyl residue in each peptide, and the intensity decay of the Asu1,6-analogues in which the disulfide bridge is substituted by a CH2-CH2 bridge. Viscosity-dependent steady state and intensity decay measurements indicated that fluorescence resonance energy transfer (FRET) from tyrosyl phenol to the disulfide 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 resonance energy transfer (FRET) to the weakly absorbing disulfide bridge and a distribution of donor-to-acceptor distances. Energy transfer efficiencies were determined from both time-resolved and steady-state measurements. Fitting the frequency-domain phase and modulation data to a Gaussian distance distribution indicated that the average inter-chromophoric distance (Rav) is similar in both compounds, Rav = 7.94 A for oxytocin and Rav = 8.00 A for vasopressin. However, the width of the distance distribution is narrower for vasopression (hw = 2.80 A) than for oxytocin (hw = 3.58 A), which is consistent with restriction of the tyrosine phenol motion due to its stacking wih the Phe3 side chain of vasopressin. Finally, the recovered distance distribution functions are compared with histograms describing the distance between the chromophores during the course of long, in vacuo, molecular dynamics runs using the computer program CHARMm and the QUANTA 3.0 parameters.
Szmacinski
H
H
Department of Biological Chemistry, Center for Fluorescence Spectroscopy, University of Maryland at Baltimore 21201, USA.
Wiczk
W
W
Fishman
M N
MN
Eis
P S
PS
Lakowicz
J R
JR
Johnson
M L
ML
D000595:Amino Acid Sequence; D001127:Arginine Vasopressin / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004220:Disulfides / Q000737:chemistry*; D007700:Kinetics; D010121:Oxytocin / Q000737:chemistry*; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry*
D004220:Disulfides; D001127:Arginine Vasopressin; D014443:Tyrosine; D010121:Oxytocin