Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer

Abstract

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.

Cyclic Peptides Bearing a Side‐Chain Tail: A Tool to Model the Structure and Reactivity of Protein Zinc Sites

“…Fluorescence kinetic measurements allowed us to monitor the destruction of the Zn(Cys) 4 center and the formation of disulfides, which are efficient quenchers of tyrosine fluorescence. [19] The kinetic traces were perfectly fitted with a single exponential. The apparent pseudo-first-order constant k obs is proportional to the concentration of H 2 O 2 .…”

Influence of solvent and configuration of residues at positions 2 and 3 on distance and mobility of pharmacophore groups at positions 1 and 4 in cyclic enkephalin analogues

“…Simultaneously, the parameters of the Gaussian become poorly determinable, which prevented us from introducing more Gaussian components. We therefore decided to keep the single‐Gaussian approximation, as in many related studies6, 15, 16, 18–22, 28–32, 35, 36, 39–43. The greatest average distances ( r av ) between chromophores are observed for enkephalin analogues dissolved in DMSO, whereas the smallest in water {except F(NO 2 )‐[ D,D ‐EN]}.…”

“…According to the literature46, 53 for distances lower than 10 Å the energy transfer according to the Dexter mechanism proceeds more efficiently than that based on the dipole–dipole interactions. The Förster model was successfully applied in the studies the distance and distance distribution in small peptides, in which the energy donor and acceptor were separated by few amino acid residues 6, 10–17, 21–23. Also, in the pioneering work of Stryer and Haugland, in which the correctness of the Förster model was verified, the donor was separated from the acceptor by 1–12 proline residues 54.…”

“…The calculation of distances also requires the knowledge of κ 2 . In our paper we assume κ 2 = ⅔ as in the work of Szmacinski et al21 and Wiczk et al22 Such an assumption is plausible because both chromophores can freely rotate about the C α C β and C β C γ bonds, which should lead to averaging the orientation factor 44–46. There are also previously published data about the relation between κ 2 and the parameters of the distance distribution showing that the parameters calculated with κ 2 = ⅔ and κ 2 = 2 differ at most by several percent40, 45 which is, in our case, within experimental error.…”

“…Time‐resolved data can be used to recover the conformational distribution or distance distribution between the donor and acceptor as well as the mobility of side chains and peptide backbone. Time‐resolved fluorescence measurements have been used to recover the distance distributions in peptides,6, 13–23 glycopeptides,24–27 proteins,28–38 and DNA 39–43…”