Characterization of the human calmodulin-like protein expressed in Escherichia coli

Rhyner, Johannes A.; Koller, Markus; Durussel-Gerber, Isabelle; Cox, Jos A.; Strehler, Emanuel E.

doi:10.1021/bi00166a017

Cited by 67 publications

(70 citation statements)

References 54 publications

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“…Bradford protein assay kits were from Bio-Rad. Recombinant human CaM was purified from Escherichia coli by published procedures (28). DNA purification kits and Ni 2ϩ -NTA agarose were purchased from QIAGEN (Chatsworth, CA).…”

Section: Methodsmentioning

confidence: 99%

Electron Transfer and Catalytic Activity of Nitric Oxide Synthases

Nishida

Montellano

1998

Journal of Biological Chemistry

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The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of ⅐NO synthesis, cytochrome c reduction, and NADPH utilization and in the Ca 2؉ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of ⅐NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca The flavin domain uncouples the electrons provided by NADPH and delivers them, one at a time, to the prosthetic heme iron atom. H 4 B is required for ⅐NO synthesis by all three proteins, but its role in the catalytic process remains unclear (9).Clear differences exist in the Ca 2ϩ and CaM dependence of the three NOS isoforms. The binding of CaM to nNOS and eNOS, the two constitutive isoforms, is Ca 2ϩ -dependent and reversible (10, 11), whereas the binding of CaM to iNOS is essentially a Ca 2ϩ -independent, irreversible process (12). Thus, the catalytic activities of nNOS and eNOS are regulated by cellular Ca 2ϩ -levels, whereas the activity of iNOS is insensitive to the Ca 2ϩ concentration and is primarily regulated by the rate at which the protein is synthesized (12). In addition to the heme, CaM, and reductase domains common to all the isoforms, nNOS has an additional N-terminal domain thought to be involved in subcellular targeting (13,14), and eNOS is unique in that it has myristoylation and palmitoylation sites that target it to the membrane (15, 16).The catalytic activities of the NOS isoforms differ considerably, regardless of whether activity is measured as ⅐NO formation or cytochrome c reduction. Regardless of which of these two parameters is measured, the activities of iNOS and nNOS are considerably higher than that of eNOS. Thus, in our hands, the V max values for ⅐NO formation by recombinant iNOS (17), nNOS (18), and eNOS (19) were found to be ϳ800, ϳ400, and ϳ130 nmol min Ϫ1 mg Ϫ1 , respectively, and the rates of electron transfer to cytochrome c in the presence of CaM and Ca 2ϩ were ϳ45,000, ϳ44,000, and ϳ1,800 nmol min Ϫ1 mg Ϫ1 , respectively. In each case, the reductase domain was able to transfer electrons to cytochrome c at a rate much greater than the maximum rate of ⅐NO production, although the nature of the ratelimiting step(s) in ⅐NO formation and the reason(s) for the differences in the catalytic activities of the isoforms are unclear. The reduction of cytochrome c by nNOS and eN...

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Section: Methodsmentioning

confidence: 99%

Electron Transfer and Catalytic Activity of Nitric Oxide Synthases

Nishida

Montellano

1998

Journal of Biological Chemistry

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“…Like calmodulin (CaM), CLP contains four "EF-hand" Ca 2ϩ binding motifs but has an overall Ca 2ϩ binding affinity that is ϳ10-fold lower than in CaM (4). The x-ray crystal structure indicates that Ca 2ϩ -CLP is very similar in overall shape to Ca 2ϩ -CaM, although a notable difference is observed in the angle of the central helix with respect to the N-and C-terminal lobes.…”

mentioning

confidence: 99%

“…Accordingly, although CLP can substitute for CaM in binding and activation of some targets (e.g. CaM kinase II), many CaM targets either bind CLP with reduced affinity or not at all (4,6). On the other hand, recent data have shown that CLP interacts with unique targets, specifically with the unconventional myosin Myo-10 (7).…”

mentioning

confidence: 99%

Calmodulin-like Protein Increases Filopodia-dependent Cell Motility via Up-regulation of Myosin-10

Bennett

Mauer

Strehler

2007

Journal of Biological Chemistry

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“…2+ -Dependent Electrophoretic Shift Assay The SDS-PAGE electrophoretic mobility assay was carried out according to published protocols (11,12) . Briefly, samples of pure WT CaM, ADA-labeled engineered CaM proteins, and lysozyme were prepared at equal concentrations, and 10 µg of each protein was loaded on 4-12% gradient gels.…”

Section: Camentioning

confidence: 99%

Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications

Kulkarni

Fraseur

et al. 2015

Bioconjugate Chem.

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Calmodulin (CaM) is a widely studied Ca 2+ binding protein that is highly conserved across species and involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. To facilitate biophysical studies of CaM, researchers have tagged and mutated CaM at various sites, enabling its conjugation to fluorophores, microarrays, and other reactive partners. However, previous attempts to add a reactive label to CaM for downstream studies have generally employed nonselective labeling methods or resulted in diminished CaM function. Here we report the first engineered CaM protein that undergoes site-specific and bioorthogonal labeling while retaining wild-type activity levels. By employing a chemoenzymatic labeling approach, we achieved selective and quantitative labeling of the engineered CaM protein with an N-terminal 12-azidododecanoic acid tag; notably, addition of the tag did not interfere with the ability of CaM to bind Ca 2+ or a partner protein. The specificity of our chemoenzymatic labeling approach also allowed for selective conjugation of CaM to reactive partners in bacterial cell lysates, without intermediate purification of the engineered protein. Additionally, we prepared CaM-affinity resins that were highly effective in purifying a representative CaM-binding protein, demonstrating that the engineered CaM remains active even after surface capture. Beyond studies of CaM and CaM-binding proteins, the protein engineering and surface capture methods described here should be translatable to other proteins and other bioconjugation applications.

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Characterization of the human calmodulin-like protein expressed in Escherichia coli

Cited by 67 publications

References 54 publications

Electron Transfer and Catalytic Activity of Nitric Oxide Synthases

Electron Transfer and Catalytic Activity of Nitric Oxide Synthases

Calmodulin-like Protein Increases Filopodia-dependent Cell Motility via Up-regulation of Myosin-10

Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications

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