May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Structural Dynamics of Visual Arrestin–Rhodopsin Binding
Author Affiliations & Notes
  • S.A. Vishnivetskiy
    Pharmacology, Vanderbilt University, Nashville, TN
  • S.M. Hanson
    Pharmacology, Vanderbilt University, Nashville, TN
  • D.J. Francis
    Biophysics, Medical College of Wisconsin, Milwaukee, WI
  • W.L. Hubbell
    JSEI, UCLA, Los Angeles, CA
  • C.S. Klug
    Biophysics, Medical College of Wisconsin, Milwaukee, WI
  • V.V. Gurevich
    Pharmacology, Vanderbilt University, Nashville, TN
  • Footnotes
    Commercial Relationships  S.A. Vishnivetskiy, None; S.M. Hanson, None; D.J. Francis, None; W.L. Hubbell, None; C.S. Klug, None; V.V. Gurevich, None.
  • Footnotes
    Support  NIH grants EY11500, GM63097, AI58024, GM 70642, EY00331, EY05216, GM07628
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 825. doi:
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      S.A. Vishnivetskiy, S.M. Hanson, D.J. Francis, W.L. Hubbell, C.S. Klug, V.V. Gurevich; Structural Dynamics of Visual Arrestin–Rhodopsin Binding . Invest. Ophthalmol. Vis. Sci. 2006;47(13):825.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Intracellular signaling by light–activated rhodopsin (Rh*) is rapidly quenched by its phosphorylation and subsequent binding of visual arrestin. Numerous lines of indirect evidence suggest that arrestin changes its conformation upon binding to phosphorylated light–activated rhodopsin (P–Rh*). To directly elucidate the mechanism of arrestin transition into its active state we employed site direct spin labeling (SDSL) –EPR.

Methods: : Cysteines were introduced in the three elements participating in one of the intramolecular interactions in free arrestin. These mutants were spin–labeled, and the mobility of the side chain was measured in solution and bound to the different functional forms of rhodopsin.

Results: : Arrestin mutants with label at positions 12 and 16 (ß–strand I), 103 and 111 (α–helix I), 376 and 381 (C–tail) were tested. The mobility of the label in both C–tail positions increases and in the α–helix decreases upon binding to dark P–Rh. The mobility in both regions did not change further upon light–activation. Very small or no changes were observed for ß–strand I mutants under similar experimental conditions. Several doubly–labeled mutants were created to determine distances between structural elements in free and P–Rh–bound arrestin. The distance between 16 and 381 in the basal conformation is 11–17 A, in agreement with the crystal structure. When arrestin is bound to dark P–Rh or P–Rh*, the distance between these two positions becomes greater than 20A. Similar results were obtained with a mutant that was doubly–labeled in the C–tail (376) and α–helix I (103).

Conclusions: : Using SDSL–EPR we were able to monitor the structural changes in arrestin in the process of its transition into the active receptor–bound state. We found that upon binding to phosphorylated rhodopsin the arrestin C–tail moves away from both α–helix I and ß–strand I. These data provide the first direct evidence for the receptor binding–induced release of the arrestin C–tail. Arrestin interaction with phosphorylated rhodopsin regardless of its activation is sufficient for this conformational change.

Keywords: photoreceptors • protein structure/function • signal transduction 

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