N-terminal Myristoylation Regulates Calcium-induced Conformational Changes in Neuronal Calcium Sensor-1

Jeromin, Andreas; Dasari, Muralidhar; Parameswaran, Malavika Nair; Roder, John; Fairwell, T; Scarlata, Suzanne; Dowal, Louisa; Mustafi, Sourajit M.; Chary, Kandala V. R.; Sharma, Yogendra

doi:10.1074/jbc.m312172200

Cited by 48 publications

(66 citation statements)

References 38 publications

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“…Furthermore, the NCS-1 E120Q protein is a considerably less efficient binder of calcium since a much higher protein concentration (110 µM) is required to decrease this ratio. These results are consistent with a number of previous studies of the calcium binding properties for these variants [5,[12][13]17], and demonstrate the presence of the C-terminal 6X His-tag does not obscure calcium binding properties of the protein. Finally, even at concentrations of 220 µM the NCS-1 EF construct shows no capacity to affect the fluorescent properties of indo-1 in the presence of calcium, indicating that under these conditions it does not appreciably bind calcium.…”

Section: Calcium Binding Of Ncs-1 Variantssupporting

confidence: 92%

“…Calcium binding constants similar to those previously published [17,32] have been observed (data not shown), demonstrating there is no change in the calcium binding behavior of these constructs.…”

Section: Calcium Binding Of Ncs-1 Variantssupporting

confidence: 86%

“…In vitro, NCS-1 and myristoylated NCS-1 have been shown to preferentially bind to POPC or POPS vesicles, respectively [17]. As demonstrated in Figure 6a, nonmyristoylated NCS-1 binds POPC vesicles with an apparent K d of 31 ± 4 µM (R 2 = 0.98755) as measured by the quenching of intrinsic protein fluorescence by increasing concentrations of lipid.…”

Section: Interaction Of 6x His-tagged Ncs-1 With Lipid and Protein Bimentioning

confidence: 92%

“…The ability of myristoylated and nonmyristoylated NCS-1 to bind to large unilamellar lipid vesicles composed of palmitoyl oleoyl phosphatidyl choline (POPC) or palmitoyl oleoyl phosphatidyl serine (POPS) was assayed using intrinsic protein fluorescence, as described previously [17,21]. POPC and POPS vesicles were prepared by extrusion through a 100 nm filter (Whatman).…”

Section: Large Unilamellar Lipid Vesicle Bindingmentioning

confidence: 99%

“…Myristoylation mediates structural changes in NCS-1 that increase its stability and affect the unfolding pathway of the protein [16]. Furthermore, the addition of the Nterminal myristoyl moiety alters the affinity of NCS-1 for calcium; myristoylated and unmodified NCS-1 show markedly different calcium binding profiles and stoichiometries [6,17]. The mechanism through which myristoylation causes these larger structural effects is not currently understood.…”

Section: Introductionmentioning

confidence: 99%

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Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Cotiis

Woll

Fox

et al. 2008

Protein Expression and Purification

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We have developed a protocol to produce large quantities of high purity myristoylated and nonmyristoylated Neuronal Calcium Sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6X His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100 -200 µM). With optimized growth conditions and a high capacity metal affinity purification scheme, > 50 mg of homogenous myristoylated NCS-1 is obtained from 1 L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 EF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.

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Section: Calcium Binding Of Ncs-1 Variantssupporting

confidence: 92%

Section: Calcium Binding Of Ncs-1 Variantssupporting

confidence: 86%

Section: Interaction Of 6x His-tagged Ncs-1 With Lipid and Protein Bimentioning

confidence: 92%

Section: Large Unilamellar Lipid Vesicle Bindingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Cotiis

Woll

Fox

et al. 2008

Protein Expression and Purification

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Survey of the year 2004: literature on applications of isothermal titration calorimetry

Ababou

Ladbury

2005

J of Molecular Recognition

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The market for commercially available isothermal titration calorimeters continues to grow as new applications and methodologies are developed. Concomitantly the number of users (and abusers) increases dramatically, resulting in a steady increase in the number of publications in which isothermal titration calorimetry (ITC) plays a role. In the present review, we will focus on areas where ITC is making a significant contribution and will highlight some interesting applications of the technique. This overview of papers published in 2004 also discusses current issues of interest in the development of ITC as a tool of choice in the determination of the thermodynamics of molecular recognition and interaction.

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Tandem Repetitive D Domains of the Sperm Ligand Zonadhesin Evolve Faster in the Paralogue Than in the Orthologue Comparison

Herlyn

Zischler

2006

J Mol Evol

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Gene duplication is regarded as an important evolutionary mechanism creating genetic and phenotypic novelty. At the same time, the evolutionary mechanisms following gene duplication have been a subject of much debate. Here we analyze the sequence evolution of zonadhesin, a mammalian sperm ligand that binds to the oocyte zona pellucida in a species-specific manner. In pig, rabbit, and primates, precursor zonadhesin comprises, among others, one partial and four complete tandem repetitive D domains. The mouse precursor is distinguished by 20 additional partial D3 domains consisting of 120 amino acids each. This gene structure allows sequence comparison in both paralogues and orthologues. Detailed sequence analysis reveals that D domains evolve faster across paralogues than orthologues. Moreover, at the codon level, partial D3 paralogues of mouse show evidence of positive selection, whereas the corresponding orthologues do not. Individual posttranslational motif patterns and positive selection point to neofunctionalization of partial D3 paralogues of mouse, rather than subfunctionalization. However, as we found additional evidence for homogenization by partial gene conversion, sequence evolution of partial D3 paralogues of mouse might be better described as a combination of divergent and convergent evolution. So far, the divergence at the codon level has outbalanced the convergence at the level of smaller fragments. The probable driving force behind the evolutionary patterns observed is sexual selection. We finally discuss whether the functional determination influences the evolutionary regime acting on sperm ligands and egg receptors, respectively.

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N-terminal Myristoylation Regulates Calcium-induced Conformational Changes in Neuronal Calcium Sensor-1

Cited by 48 publications

References 38 publications

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Survey of the year 2004: literature on applications of isothermal titration calorimetry

Tandem Repetitive D Domains of the Sperm Ligand Zonadhesin Evolve Faster in the Paralogue Than in the Orthologue Comparison

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