Perturbations of the local environment of the single tyrosine residue in neurophysin upon ligand binding as determined by steady-state and time-resolved fluorescence quenching

摘要

The neurophysins (NP) are proteins which function in neurohypophyseal peptide hormone transport and storage. We have been investigating the fluorescence properties of the single tyrosine residue (Tyr$+49$/) of bovine NPs. Since the intensity of Tyr$+49$/ fluorescence increases on ligand binding, measurements have been made with and without small peptides which bind at the hormone binding site. Steady-state iodide quenching produces Stern-Volmer plots with downward and upward curvature for NP and the NP:Phe-PheNH$-2$/ complex, respectively. Non- linear Stern-Volmer plots can result from multiple conformations of the solvated protein, multiple environments for the rotational isomers of the phenol side chain (rotamers), as well as from dynamic and static interactions of Tyr$+49$/ with the quencher. To help explain the steady-state data, time-resolved fluorescence quenching studies have been performed. Both NP and the NP:Phe-PheNH$-2$/ complex exhibit fluorescence intensity decays that can be fit by the sum of three exponentials that consist of the same time constants but with different weighting (amplitude) terms. With increasing $LB@I$+$MIN$/$RB@, the decay parameters for Tyr$+49$/ in NP are consistent with a multiple species model due to rotamers of Tyr$+49$/, the amplitudes are quencher independent and the lifetimes result in linear Stern-Volmer plots. The quenching studies on the NP:PhePheNH$-2$/ binary complex, however, cannot be analyzed in terms of a specific model because the fractional intensities of two of the exponential components are small.