Regulatory and Structural EF-Hand Motifs of Neuronal Calcium Sensor-1: Mg2+ Modulates Ca2+ Binding, Ca2+-Induced Conformational Changes, and Equilibrium Unfolding Transitions

Penmatsa, Aravind; Chandra, Kousik; Reddy, Pasham Parameshwar; Jeromin, Andreas; Chary, Kandala V. R.; Sharma, Yogendra

doi:10.1016/j.jmb.2007.12.033

Cited by 75 publications

(116 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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 the presence of calcium, the spectra shows a dispersion of resonances along both frequency axes indicating the protein construct containing the 6X His-tag is properly folded and is of suitable quality for structural study by NMR based techniques ( Figure 5). Comparison of this spectrum with a recently published HSQC spectrum of a myristoylated NCS-1 construct lacking a 6X His-tag [32] demonstrates a very similar cross-peak profile suggesting the proteins have a common tertiary structure. Furthermore, both spectra also demonstrate a shift in cross-peak profile following the addition of calcium.…”

Section: Effect Of the C-terminal 6x His-tag And Ef-hand Point Mutatimentioning

confidence: 62%

See 1 more Smart Citation

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

Cotiis

Woll

Fox

et al. 2008

Protein Expression and Purification

View full text Add to dashboard Cite

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.

show abstract

Section: Calcium Binding Of Ncs-1 Variantssupporting

confidence: 86%

Section: Effect Of the C-terminal 6x His-tag And Ef-hand Point Mutatimentioning

confidence: 62%

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

Cotiis

Woll

Fox

et al. 2008

Protein Expression and Purification

View full text Add to dashboard Cite

show abstract

“…This may be important in the prevention of nonspecific interactions in the absence of a specific Ca 2þ -signal. In the presence of elevated [Ca 2þ ] i , EF2 and EF3 become Ca 2þ -occupied, simultaneously followed by Ca 2þ binding to EF4 (Aravind et al 2008). The Mg þþ bound form of NCS-1 has a fivefold lower affinity for Ca 2þ than the Mg þþ -free/ Ca 2þ -free apo-form.…”

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 99%

“…The Mg þþ bound form of NCS-1 has a fivefold lower affinity for Ca 2þ than the Mg þþ -free/ Ca 2þ -free apo-form. This implies that Mg þþ binding permits significant modulation of NCS-1 and is important in fine tuning its Ca 2þ -sensing properties (Aravind et al 2008;Mikhaylova et al 2009). …”

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 99%

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

McCue

Haynes²,

Burgoyne

2010

Cold Spring Harbor Perspectives in Biology

View full text Add to dashboard Cite

“…Reversible binding of calcium to EF-hand proteins enables proteins of this group to participate in biological functions such as calcium transduction and signal modulation. 37,38 In this study, we computationally transfer one of the non-EF-hand calcium-binding pockets found in thermitase protein (PDB: 1thm). This protein contains three calcium-binding sites, and all three contribute to the protein's thermal stability.…”

Section: Optgraft Predictionsmentioning

confidence: 99%

OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

2008

View full text Add to dashboard Cite

One of the many challenging tasks of protein design is the introduction of a completely new function into an existing protein scaffold. In this study, we introduce a new computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. This is accomplished by introducing a two-level procedure where we first identify where are the most appropriate locations to graft the new binding pocket into the protein fold by minimizing the departure from a set of geometric restraints using mixed-integer linear optimization. On identifying the suitable locations that can accommodate the new binding pocket, CHARMM energy calculations are employed to identify what mutations in the neighboring residues, if any, are needed to ensure that the minimum energy conformation of the binding pocket conserves the desired geometry. This computational framework is benchmarked against the results available in the literature for engineering a copper binding site into thioredoxin protein. Subsequently, OptGraft is used to guide the transfer of a calcium-binding pocket from thermitase protein (PDB: 1thm) into the first domain of CD2 protein (PDB:1hng). Experimental characterization of three de novo redesigned proteins with grafted calcium-binding centers demonstrated that they all exhibit high affinities for terbium (K d $ 22, 38, and 55 lM) and can selectively bind calcium over magnesium.

show abstract

Regulatory and Structural EF-Hand Motifs of Neuronal Calcium Sensor-1: Mg2+ Modulates Ca2+ Binding, Ca2+-Induced Conformational Changes, and Equilibrium Unfolding Transitions

Cited by 75 publications

References 50 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

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

Contact Info

Product

Resources

About