The long-term goal of this work is to elucidate molecular mechanisms of vertebrate photoresponse signal transduction. In the current application we propose experiments to characterize the interaction of recoverin with rhodopsin kinase. This proposal is based upon preliminary experiments from the last grant period in which a previously unknown conformation of recoverin was formed when Ca2+-activated recoverin was bound to either the full RGS domain of rhodopsin kinase or an N-terminal peptide fragment of the kinase. Importantly, this new conformation appears to be present as a minor species (RvCaB) in an equilibrium mixture (RvCaA/RvCaB) formed upon the binding of Ca2+ to recoverin even in the absence of the kinase. Our approach will be to use a combination of biophysical techniques (primarily NMR spectroscopy) and biochemical analyses with purified components under in vitro conditions to unravel details of both the structure and mechanism of action for the interaction of these two proteins. There are three Specific Aims in this proposal: 1. To complete structural characterization of RvCaB;2. To probe through mutagenesis studies which amino acid residues in recovern contribue to stabilization of RvCaB;and 3. To determine in atomic detail the structure of the recoverin/rhodopsin kinase complex with initial focus on the RGS domain of the kinase.

Public Health Relevance

This study will have an impact in three areas: 1. Understanding fundamental mechanisms of signaling molecules involved in protein-protein interactions;2. Understanding of aberrant recoverin expression for cancer-associated retinopathy and cell proliferation;and 3. Understanding general mechanisms of neuronal calcium sensor proteins.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biology and Diseases of the Posterior Eye Study Section (BDPE)
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Neuhold, Lisa
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Brandeis University
Schools of Arts and Sciences
United States
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Chakrabarti, Kalyan S; Agafonov, Roman V; Pontiggia, Francesco et al. (2016) Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis. Cell Rep 14:32-42
Kumar, Ramasamy P; Ranaghan, Matthew J; Ganjei, Allen Y et al. (2015) Crystal Structure of Recoverin with Calcium Ions Bound to Both Functional EF Hands. Biochemistry 54:7222-8
Ranaghan, Matthew J; Kumar, Ramasamy P; Chakrabarti, Kalyan S et al. (2013) A highly conserved cysteine of neuronal calcium-sensing proteins controls cooperative binding of Ca2+ to recoverin. J Biol Chem 288:36160-7
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