Related ArticlesTwo-dimensional NMR studies of selenomethionyl calmodulin.
J Mol Biol. 1994 Jun 17;239(4):545-54
Authors: Zhang M, Vogel HJ
Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins. The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on Ca(2+)-CaM; these two regions are very rich in Met residues. To obtain more information about the role of these residues, we have biosynthetically incorporated selenomethionine (SeMet) in place of the nine Met residues of CaM. Amino acid analysis shows that the SeMet-CaM contains 15% Met and 85% SeMet. SeMet-CaM retains many of the properties of the wild-type protein; it activates the enzyme cyclic nucleotide phosphodiesterase, it binds to phenyl-Sepharose and myosin light chain kinase (MLCK) in a calcium-dependent manner, and it experiences a calcium-dependent band shift during SDS-gel electrophoresis. Moreover, by comparing the natural abundance (1H,13C)-heteronuclear multiple quantum coherence (HMQC) spectra of the calcium, apo and target peptide-bound forms of wild-type CaM and SeMet-CaM, we have found that the two proteins have very similar, if not identical, structures. Thus, the substitution of SeMet for Met does not cause a change in the conformation and function of CaM, in agreement with the results obtained for other proteins. The apo, calcium and target peptide-bound forms of SeMet-CaM were subsequently studied by natural abundance (1H,77Se)-heteronuclear multiple bond correlation (HMBC) and (1H,13C)-HMQC NMR. Nine well-resolved 77Se resonances could be observed. Substitution of SeMet for Met gave rise to the same 1H and 13C chemical shift changes for each individual Met residue, this facilitated making the assignments from known 1H,13C assignments of the Met residues. Some of these assignments were confirmed by studying Met-->Leu mutants of CaM. With the exception of Met76, which always remains solvent exposed, all resonances experienced large 77Se chemical shift changes upon the addition of Ca2+ and the MLCK peptide. The large shift changes indicate that the electron distribution in the SeMet side-chain can be adjusted for the different states of CaM, suggesting that the polarizability of sulfur or selenium may be important for the proper functioning of CaM. This study also shows that the natural abundance (1H,77Se)-HMBC experiment provides a sensitive approach for the study of SeMet proteins.
[NMR paper] Metal ion binding to calmodulin: NMR and fluorescence studies.
Metal ion binding to calmodulin: NMR and fluorescence studies.
Related Articles Metal ion binding to calmodulin: NMR and fluorescence studies.
Biometals. 1998 Sep;11(3):213-22
Authors: Ouyang H, Vogel HJ
Calmodulin is an important second messenger protein which is involved in a large variety of cellular pathways. Calmodulin is sensitive to fluctuations in the intracellular Ca2+ levels and is activated by the binding of four Ca2+ ions. In spite of the important role it plays in signal transduction pathways, it shows a surprisingly broad...
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[NMR paper] NMR studies of caldesmon-calmodulin interactions.
NMR studies of caldesmon-calmodulin interactions.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles NMR studies of caldesmon-calmodulin interactions.
Biochemistry. 1997 Mar 11;36(10):2817-25
Authors: Zhou N, Yuan T, Mak AS, Vogel HJ
The binding of the calcium-regulatory protein calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. Two regions of caldesmon have been identified as putative calmodulin-binding domains. We have earlier reported on the...
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[NMR paper] NMR studies of caldesmon-calmodulin interactions.
NMR studies of caldesmon-calmodulin interactions.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles NMR studies of caldesmon-calmodulin interactions.
Biochemistry. 1997 Mar 11;36(10):2817-25
Authors: Zhou N, Yuan T, Mak AS, Vogel HJ
The binding of the calcium-regulatory protein calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. Two regions of caldesmon have been identified as putative calmodulin-binding domains. We have earlier reported on the...
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08-22-2010 03:03 PM
[NMR paper] Protein engineering and NMR studies of calmodulin.
Protein engineering and NMR studies of calmodulin.
Related Articles Protein engineering and NMR studies of calmodulin.
Mol Cell Biochem. 1995 Aug-Sep;149-150:3-15
Authors: Vogel HJ, Zhang M
The calcium regulatory protein calmodulin (CaM) plays a role as an on-off switch in the activation of many enzymes and proteins. CaM has a dumbbell shaped structure with two folded domains, which are connected by a flexible linker in solution. The calmodulin-binding domains of the target proteins are contained in 20 residue long amino acid sequences, that...
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[NMR paper] NMR studies of the methionine methyl groups in calmodulin.
NMR studies of the methionine methyl groups in calmodulin.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles NMR studies of the methionine methyl groups in calmodulin.
FEBS Lett. 1995 Jun 12;366(2-3):104-8
Authors: Siivari K, Zhang M, Palmer AG, Vogel HJ
Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein that can regulate a wide variety of cellular events. The protein contains 9 Met out of a total of 148 amino acid residues. The binding of Ca2+ to CaM induces...
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08-22-2010 03:41 AM
[NMR paper] Two-dimensional NMR studies of selenomethionyl calmodulin.
Two-dimensional NMR studies of selenomethionyl calmodulin.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Two-dimensional NMR studies of selenomethionyl calmodulin.
J Mol Biol. 1994 Jun 17;239(4):545-54
Authors: Zhang M, Vogel HJ
Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins. The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on...
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08-22-2010 03:33 AM
[NMR paper] Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H
Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy.
Related Articles Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy.
Biochim Biophys Acta. 1992 Nov 10;1160(1):22-34
Authors: Gao Y, Levine BA, Mornet D, Slatter DA, Strasburg GM
In order to identify comparative aspects of the interaction of calmodulin with its target proteins, proton magnetic-resonance studies of complex formation between calmodulin and defined segments of phospholamban...
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08-21-2010 11:45 PM
[NMR paper] Purification of cloned trypanosomal calmodulin and preliminary NMR studies.
Purification of cloned trypanosomal calmodulin and preliminary NMR studies.
Related Articles Purification of cloned trypanosomal calmodulin and preliminary NMR studies.
J Chromatogr. 1991 Feb 22;539(2):501-5
Authors: Sweeney PJ, Walker JM, Reid DG, Elshourbagy N
Cloned trypanosomal calmodulin was expressed in Escherichia coli and purified to homogeneity using hydrophobic interaction chromatography on phenyl-Sepharose. The purified protein was subjected to NMR analysis which allows detailed changes to be observed when, firstly, calcium, and...