Dataset Information

Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin.

ABSTRACT: Arrestins regulate signaling and trafficking of G protein-coupled receptors by virtue of their preferential binding to the phosphorylated active form of the receptor. To identify sites in arrestin involved in receptor interaction, a nitroxide-containing side chain was introduced at each of 28 different positions in visual arrestin, and the dynamics of the side chain was used to monitor arrestin interaction with phosphorylated forms of its cognate receptor, rhodopsin. At physiological concentrations, visual arrestin associates with both inactive dark phosphorylated rhodopsin (P-Rh) and light-activated phosphorylated rhodopsin (P-Rh*). Residues distributed over the concave surfaces of the two arrestin domains are involved in weak interactions with both states of phosphorhodopsin, and the flexible C-terminal sequence (C-tail) of arrestin becomes dynamically disordered in both complexes. A large-scale movement of the C-tail is demonstrated by direct distance measurements using a doubly labeled arrestin with one nitroxide in the C-tail and the other in the N-domain. Despite some overlap, the molecular "footprint" of arrestin bound to P-Rh and P-Rh* is different, showing the structure of the complexes to be unique. Strong immobilizing interactions with residues in a highly flexible loop between beta-strands V and VI are only observed in complex with the activated state. This result identifies this loop as a key recognition site in the arrestin-P-Rh* complex and supports the view that flexible sequences are key elements in protein-protein interactions.

SUBMITTER: Hanson SM

PROVIDER: S-EPMC1458767 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin.

Hanson Susan M SM Francis Derek J DJ Vishnivetskiy Sergey A SA Kolobova Elena A EA Hubbell Wayne L WL Klug Candice S CS Gurevich Vsevolod V VV

Proceedings of the National Academy of Sciences of the United States of America 20060317 13

Arrestins regulate signaling and trafficking of G protein-coupled receptors by virtue of their preferential binding to the phosphorylated active form of the receptor. To identify sites in arrestin involved in receptor interaction, a nitroxide-containing side chain was introduced at each of 28 different positions in visual arrestin, and the dynamics of the side chain was used to monitor arrestin interaction with phosphorylated forms of its cognate receptor, rhodopsin. At physiological concentrati ...[more]

PMID: 16547131

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Project description:An extensive set of electron spin resonance spectra was obtained over a wide range of frequencies (9, 95, 170, and 240 GHz) and temperatures (2 to 32 degrees C) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model, the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2 are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered.

Dataset Information

Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin.

Publications

Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin.

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