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Double Electron-Electron Resonance Probes Ca2+-Induced Conformational Changes and Dimerization of Recoverin

Myers, William K.; Xu, Xianzhong; Li, Congmin; Lagerstedt, Jens LU ; Budamagunta, Madhu S.; Voss, John C.; Britt, R. David and Ames, James B. (2013) In Biochemistry 52(34). p.5800-5808
Abstract
Recoverin a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca2+-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca2+-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron electron resonance (DEER) experiments on recoverin that probe Ca2+-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and... (More)
Recoverin a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca2+-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca2+-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron electron resonance (DEER) experiments on recoverin that probe Ca2+-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 +/- 0.1 nm for Ca2+-free recoverin and 3.7 +/- 0.1 nm for Ca2+-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca2+-bound recoverin may represent an intermolecular distance between C39 and N120. N-15 NMR relaxation analysis and CW-EPR experiments both confirm that Ca2+-bound recoverin forms a dimer at protein concentrations above 100 mu M, whereas Ca2+-free recoverin is monomeric We propose that Ca2+-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca2+-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca2+-induced dimerization is functionally important but not well understood. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
52
issue
34
pages
5800 - 5808
publisher
The American Chemical Society (ACS)
external identifiers
  • wos:000323810900010
  • scopus:84883238526
ISSN
0006-2960
DOI
10.1021/bi400538w
language
English
LU publication?
yes
id
4eabec65-41e0-4828-aa8b-c93e7ecaf245 (old id 4062932)
date added to LUP
2014-03-03 08:11:42
date last changed
2019-02-20 01:41:24
@article{4eabec65-41e0-4828-aa8b-c93e7ecaf245,
  abstract     = {Recoverin a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca2+-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca2+-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron electron resonance (DEER) experiments on recoverin that probe Ca2+-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 +/- 0.1 nm for Ca2+-free recoverin and 3.7 +/- 0.1 nm for Ca2+-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca2+-bound recoverin may represent an intermolecular distance between C39 and N120. N-15 NMR relaxation analysis and CW-EPR experiments both confirm that Ca2+-bound recoverin forms a dimer at protein concentrations above 100 mu M, whereas Ca2+-free recoverin is monomeric We propose that Ca2+-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca2+-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca2+-induced dimerization is functionally important but not well understood.},
  author       = {Myers, William K. and Xu, Xianzhong and Li, Congmin and Lagerstedt, Jens and Budamagunta, Madhu S. and Voss, John C. and Britt, R. David and Ames, James B.},
  issn         = {0006-2960},
  language     = {eng},
  number       = {34},
  pages        = {5800--5808},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Biochemistry},
  title        = {Double Electron-Electron Resonance Probes Ca2+-Induced Conformational Changes and Dimerization of Recoverin},
  url          = {http://dx.doi.org/10.1021/bi400538w},
  volume       = {52},
  year         = {2013},
}