Calcium binding to recoverin: implications for secondary structure and membrane association.
Recoverin is an EF-hand calcium-binding protein reportedly involved in the transduction of light by vertebrate photoreceptor cells. It also is an autoantigen in a cancer-associated degenerative disease of the retina. Measurements by circular dichroism presented here demonstrate that the binding of calcium to recoverin causes large structural changes. increasing the alpha-helical content of the protein and decreasing its beta-turn, beta-sheet and 'other' structures. The maximum helical content (67%) was observed at 100 microM free calcium and, unlike calmodulin, decreased as the calcium concentration was modulated in either direction from this value. Fluorescence measurements indicated that recoverin may aggregate or undergo structural changes independent of calcium binding as the calcium concentration is increased above 100 microM. EGTA also appeared to affect the structure of recoverin independent of its chelation of calcium. While calcium-induced conformational changes have been proposed to alter the membrane binding of recoverin through association of its myristoylated amino terminus, in the experiments presented here the partitioning of recoverin between the cytoplasmic and membrane compartments of the rod photoreceptor outer segment was unaffected by the concentration of calcium, therefore it appears unlikely that a calcium-myristoyl switch acts alone to anchor recoverin directly to the membrane. These experiments were conducted with native recoverin which is heterogeneously acylated, but mass spectrometry confirmed that simple chromatographic methods could be devised to isolate the different forms of recoverin for further studies.
Johnson
W C
WC
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331, USA.
Palczewski
K
K
Gorczyca
W A
WA
Riazance-Lawrence
J H
JH
Witkowska
D
D
Polans
A S
AS
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D004533:Egtazic Acid / Q000494:pharmacology; D005136:Eye Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D008566:Membranes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D051603:Recoverin; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014443:Tyrosine
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D051603:Recoverin; D051597:Hippocalcin; D014443:Tyrosine; D004533:Egtazic Acid; D014364:Tryptophan; D002118:Calcium