Protein Engineering, Vol. 13, No. 11, 783-790,
November 2000
© 2000 Oxford University Press
Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites
1 Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Region 142292, 2 Department of Enzymology, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899 and 3 Branch of M. M. Shemyakin and Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Moscow Region 142292, Russia
A molecule of the photoreceptor Ca2+-binding protein recoverin contains four potential EF-hand Ca2+-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca2+-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca2+-binding constants according to the sequential binding scheme gave the values 3.7 x 106 and 3.1 x 105 M–1 for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  O. H. Weiergraber, I. I. Senin, E. Y. Zernii, V. A. Churumova, N. A. Kovaleva, A. A. Nazipova, S. E. Permyakov, E. A. Permyakov, P. P. Philippov, J. Granzin, et al. Tuning of a Neuronal Calcium Sensor J. Biol. Chem., December 8, 2006; 281(49): 37594 - 37602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. T. Buchanan, J. B. Ames, S. H. Asfaw, J. N. Wingard, C. L. Olson, P. T. Campana, A. P. U. Araujo, and D. M. Engman A Flagellum-specific Calcium Sensor J. Biol. Chem., December 2, 2005; 280(48): 40104 - 40111. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Chehab, O. R. Patharkar, A. D. Hegeman, T. Taybi, and J. C. Cushman Autophosphorylation and Subcellular Localization Dynamics of a Salt- and Water Deficit-Induced Calcium-Dependent Protein Kinase from Ice Plant Plant Physiology, July 1, 2004; 135(3): 1430 - 1446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. O'Callaghan, A. V. Tepikin, and R. D. Burgoyne Dynamics and calcium sensitivity of the Ca2+/myristoyl switch protein hippocalcin in living cells J. Cell Biol., November 24, 2003; 163(4): 715 - 721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. H. Weiergraber, I. I. Senin, P. P. Philippov, J. Granzin, and K.-W. Koch Impact of N-terminal Myristoylation on the Ca2+-dependent Conformational Transition in Recoverin J. Biol. Chem., June 13, 2003; 278(25): 22972 - 22979. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. I. Senin, T. Fischer, K. E. Komolov, D. V. Zinchenko, P. P. Philippov, and K.-W. Koch Ca2+-Myristoyl Switch in the Neuronal Calcium Sensor Recoverin Requires Different Functions of Ca2+-binding Sites J. Biol. Chem., December 20, 2002; 277(52): 50365 - 50372. [Abstract] [Full Text] [PDF]
|
|