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| | {{STRUCTURE_1la3| PDB=1la3 | SCENE= }} | | {{STRUCTURE_1la3| PDB=1la3 | SCENE= }} |
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| - | '''Solution structure of recoverin mutant, E85Q'''
| + | ===Solution structure of recoverin mutant, E85Q=== |
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| - | ==Overview==
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| - | Recoverin, a member of the EF-hand superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl or related fatty acyl group covalently attached to the N-terminus of recoverin facilitates the binding of recoverin to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Previous structural studies revealed that the myristoyl group of recoverin is sequestered inside the protein core in the absence of calcium. The cooperative binding of two calcium ions to the second and third EF-hands (EF-2 and EF-3) of recoverin leads to the extrusion of the fatty acid. Here we present nuclear magnetic resonance (NMR), fluorescence, and calcium-binding studies of a myristoylated recoverin mutant (myr-E85Q) designed to abolish high-affinity calcium binding to EF-2 and thereby trap the myristoylated protein with calcium bound solely to EF-3. Equilibrium calcium-binding studies confirm that only one Ca2+ binds to myr-E85Q under the conditions of this study with a dissociation constant of 100 microM. Fluorescence and NMR spectra of the Ca2+-free myr-E85Q are identical to those of Ca2+-free wild type, indicating that the E85Q mutation does not alter the stability and structure of the Ca2+-free protein. In contrast, the fluorescence and NMR spectra of half-saturated myr-E85Q (one bound Ca2+) look different from those of Ca2+-saturated wild type (two bound Ca2+), suggesting that half-saturated myr-E85Q may represent a structural intermediate. We report here the three-dimensional structure of Ca2+-bound myr-E85Q as determined by NMR spectroscopy. The N-terminal myristoyl group of Ca2+-bound myr-E85Q is sequestered within a hydrophobic cavity lined by many aromatic residues (F23, W31, Y53, F56, F83, and Y86) resembling that of Ca2+-free recoverin. The structure of Ca2+-bound myr-E85Q in the N-terminal region (residues 2-90) is similar to that of Ca2+-free recoverin, whereas the C-terminal region (residues 100-202) is more similar to that of Ca2+-bound wild type. Hence, the structure of Ca2+-bound myr-E85Q represents a hybrid between the structures of recoverin with zero and two Ca2+ bound. The binding of Ca2+ to EF-3 leads to local structural changes within the EF-hand that alter the domain interface and cause a 45 degrees swiveling of the N- and C-terminal domains, resulting in a partial unclamping of the myristoyl group. We propose that Ca2+-bound myr-E85Q may represent a stable intermediate state in the kinetic mechanism of the calcium-myristoyl switch.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_11980481}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 11980481 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_11980481}} |
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| | ==About this Structure== | | ==About this Structure== |
| - | 1LA3 is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1LA3 OCA]. | + | 1LA3 is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus]. Full experimental information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1LA3 OCA]. |
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| | ==Reference== | | ==Reference== |
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| | [[Category: Ef-hand]] | | [[Category: Ef-hand]] |
| | [[Category: Vision]] | | [[Category: Vision]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 23:42:54 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jul 2 12:05:19 2008'' |
Revision as of 09:05, 2 July 2008
Template:STRUCTURE 1la3
Solution structure of recoverin mutant, E85Q
Template:ABSTRACT PUBMED 11980481
About this Structure
1LA3 is a Single protein structure of sequence from Bos taurus. Full experimental information is available from OCA.
Reference
Structure and calcium-binding studies of a recoverin mutant (E85Q) in an allosteric intermediate state., Ames JB, Hamasaki N, Molchanova T, Biochemistry. 2002 May 7;41(18):5776-87. PMID:11980481
Page seeded by OCA on Wed Jul 2 12:05:19 2008
