Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites
| Autor: | Sergei E. Permyakov, Pavel P. Philippov, Dmitry V. Zinchenko, Vladimir N. Uversky, Ivan I. Senin, Alexandra M. Cherskaya, Eugene A. Permyakov, A. M. Alekseev, Sergey V. Shulga-Morskoy, Aminullah A. Zargarov, Valery M. Lipkin |
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| Rok vydání: | 2001 |
| Předmět: |
Circular dichroism
Protein Denaturation Hot Temperature Protein Conformation Lipoproteins chemistry.chemical_element Bioengineering Nerve Tissue Proteins Calcium Biochemistry Protein structure Recoverin Hippocalcin Binding site Eye Proteins Molecular Biology Protein secondary structure chemistry.chemical_classification biology Circular Dichroism Calcium-Binding Proteins Tryptophan Hydrogen-Ion Concentration Amino acid chemistry Microscopy Fluorescence Mutation Biophysics biology.protein Biotechnology Protein Binding |
| Zdroj: | Protein engineering. 13(11) |
| ISSN: | 0269-2139 |
| Popis: | A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-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 Ca(2+)-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 Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) 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. |
| Databáze: | OpenAIRE |
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