May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Loop Movement in Arrestin is Necessary for Binding to Light-Activated Rhodopsin
Author Affiliations & Notes
  • W. Smith
    Ophthalmology, University of Florida, Gainesville, Florida
  • M. E. Sommer
    Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon
  • D. L. Farrens
    Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon
  • J. H. McDowell
    Ophthalmology, University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships W. Smith, None; M.E. Sommer, None; D.L. Farrens, None; J.H. McDowell, None.
  • Footnotes
    Support NEI grants EY06225 and EY08571, Kirchgessner Foundation
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1111. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      W. Smith, M. E. Sommer, D. L. Farrens, J. H. McDowell; Loop Movement in Arrestin is Necessary for Binding to Light-Activated Rhodopsin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1111. doi: https://doi.org/.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose:: Studies show the loop structure containing Ile-72 (Loop V-VI) in bovine visual arrestin adopts multiple conformations in crystal structures. Previously, we have shown that spectroscopic probes in this loop show changes upon binding to light-activated phosphorhodopsin (R*P), consistent with this loop moving into close contact with rhodopsin. In this study, we have further investigated whether Loop V-VI undergoes conformational changes during binding to R*P, and if such a conformational change is necessary for binding to R*P.

Methods:: His-tagged bovine arrestin with targeted mutations was expressed in yeast and affinity purified. Arrestin was labeled with the fluorescent monobromobimane probe at I72C (=I72B) to monitor dynamic movements of the fluorescent label during binding to rhodopsin, with tryptophan residues selectively positioned to quench bimane fluorescence. Additional cysteine residues were introduced pair-wise into I72C at Glu-148, Lys-166, Lys-276, and Lys-298 to promote internal disulfide crosslinks. Arrestin was also subjected to limited trypsinolysis to further assess conformational changes during activation.

Results:: The fluorescence emission of bimane-labeled I72B arrestin was significantly quenched when either Glu-148 or Lys-298 were mutated to tryptophan, indicating close proximity of the fluorophore to these two residues. During binding to R*P, fluorescence emission was restored to near that of arrestin without the tryptophan substitutions. Substitution of both Lys-298 and Ile-72 with cysteines created an arrestin that retained arrestin’s binding selectivity for R*P in the presence of 1 mM DTT. Removal of DTT in the presence of CuSO4 to promote internal crosslinks, dramatically reduced binding to R*P. This same pattern was observed for cysteine pair I72C/E148C, but not for cysteine pairs I72C/K166C or I72C/K276C. Limited trypsinolysis showed that conformational changes induced in native arrestin by either R*P or the phosphopeptide analog of rhodopsin’s C-terminus were also blocked only in the crosslinked I72C/K298C and I72C/E148C double mutants.

Conclusions:: Our results show that during binding to R*P, Loop V-VI of arrestin moves relative to residues Glu-148 and Lys-298, which are located in the N-terminal domain. When this loop is tethered by an internal disulfide bond (either at E148C or K298C), binding to rhodopsin is blocked. These results suggest that movement of the Ile-72 loop is an important element regulating arrestin binding to rhodopsin.

Keywords: photoreceptors • protein structure/function 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×