Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM)

Rout, Ashok K.; Barnwal, Ravi Pratap; Padhan, Narendra; Bhattacharya, Alok; Chary, Kandala V. R.

doi:10.1007/s12104-008-9089-5

Cited by 3 publications

(11 citation statements)

References 13 publications

(8 reference statements)

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“…It has a well-folded N-terminal domain and an unstructured C-terminal domain. Although the CSI plot shows that the C-terminal domain has some secondary structure propensity, it does not show any tertiary fold. The observation that the C-terminal domain is unstructured is supported by low chemical shift dispersion of amide protons, insufficient NOEs, sudden disappearance of [ 15 N− 1 H] peaks in the HX experiment, negative heteronuclear 15 N− 1 H NOEs, and absence of Ca 2+ -binding sites.…”

Section: Discussionmentioning

confidence: 93%

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Calmodulin-like Protein from Entamoeba histolytica: Solution Structure and Calcium-Binding Properties of a Partially Folded Protein

et al. 2010

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The mechanism of Ca(2+)-signaling in the protozoan parasite Entamoeba histolytica is yet to be understood as many of the key regulators are still to be identified. E. histolytica encodes a number of multi-EF-hand Ca(2+)-binding proteins (EhCaBPs). Functionally only one of these molecules, EhCaBP1, has been characterized to date. The calmodulin-like protein from E. histolytica (abbreviated as EhCaM or EhCaBP3) is a 17.23 kDa monomeric protein that shows maximum sequence identity with heterologous calmodulins (CaMs). Though CaM activity has been biochemically shown in E. histolytica, there are no reports on the presence of a typical CaM. In an attempt to understand the structural and functional similarity of EhCaM with CaM, we have determined the three-dimensional (3D) solution structure of EhCaM using NMR. The EhCaM has a well-folded N-terminal domain and an unstructured C-terminal counterpart. Further, it sequentially binds only two calcium ions, an unusual mode of Ca(2+)-binding among the known CaBPs, notably both in the N-terminal domain of EhCaM. Further, EhCaM is present in the nucleus in addition to the cytoplasm as detected by immunofluorescence staining, unlike other EhCaBPs that are detected only in the cytoplasm. Therefore, this protein is likely to have a different function. The presence of unusual and a diverse set of CaBPs in E. histolytica suggests a distinct Ca(2+)-signaling process in E. histolytica. The results reported here help in understanding the structure-function relationship of CaBPs including their Ca(2+)-binding properties.

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Section: Discussionmentioning

confidence: 93%

“…The experiments recorded with the U-13 C/ 15 N doubly labeled sample for backbone and side-chain assignments were as described earlier (18). All of the spectra were processed using Felix 2002 (Accelrys Inc., San Diego, CA) and analyzed by TATAPRO (21) and Cara (22).…”

Section: Methodsmentioning

confidence: 99%

Calmodulin-like Protein from Entamoeba histolytica: Solution Structure and Calcium-Binding Properties of a Partially Folded Protein

et al. 2010

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“…The experiments with u‐ 15 N/ 13 C‐labeled protein include sensitivity‐enhanced 2D [ 15 N 1 H]‐HSQC using water‐flip‐back for minimizing the water saturation, 3D CBCANH,21 3D CBCA(CO)NH,22 3D HNCO,23 3D HN(CA)CO,24, 25 3D HNHA26, 27 and GFT (3,2)HNHA 28. NMR experiments performed for other proteins are given elsewhere 29, 30. The data transformation and processing were done on a PC using Felix 2002 software (Molecular Simulations Inc.) and analyzed using CARA 31.…”

Section: Methodsmentioning

confidence: 99%

Root‐mean‐square‐deviation‐based rapid backbone resonance assignments in proteins

Rout

Barnwal

Agarwal

et al. 2010

Magnetic Reson in Chemistry

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We have shown that the methodology based on the estimation of root-mean-square deviation (RMSD) between two sets of chemical shifts is very useful to rapidly assign the spectral signatures of (1)H(N), (13)C(α), (13)C(β), (13)C', (1)H(α) and (15)N spins of a given protein in one state from the knowledge of its resonance assignments in a different state, without resorting to routine established procedures (manual and automated). We demonstrate the utility of this methodology to rapidly assign the 3D spectra of a metal-binding protein in its holo-state from the knowledge of its assignments in apo-state, the spectra of a protein in its paramagnetic state from the knowledge of its assignments in diamagnetic state and, finally, the spectra of a mutant protein from the knowledge of the chemical shifts of the corresponding wild-type protein. The underlying assumption of this methodology is that, it is impossible for any two amino acid residues in a given protein to have all the six chemical shifts degenerate and that the protein under consideration does not undergo large conformational changes in going from one conformational state to another. The methodology has been tested using experimental data on three proteins, M-crystallin (8.5 kDa, predominantly β-sheet, for apo- to holo-state), Calbindin (7.5 kDa, predominantly α-helical, for diamagnetic to paramagnetic state and apo to holo) and EhCaBP1 (14.3 kDa, α-helical, the wild-type protein with one of its mutant). In all the cases, the extent of assignment is found to be greater than 85%.

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“…The assignment of this protein has already been published [11] and thus this served as an excellent test case. Fig.…”

Section: Application To Proteinsmentioning

confidence: 99%

“…15 N-13 C labeled holo EhCaM was obtained as a kind gift from Mr. Ashok Kumar Rout. The protein expression, purification, and assignment have been described elsewhere [11].…”

Section: Potential Applications Of (Hc)nhmentioning

confidence: 99%

Spectroscopic labeling of A, S/T in the 1H–15N HSQC spectrum of uniformly (15N–13C) labeled proteins

Chugh

Hosur

2008

Journal of Magnetic Resonance

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Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM)

Abstract: We report almost complete sequence specific 1H, 13C and 15N NMR assignments of a 151-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaM).

Cited by 3 publications

References 13 publications

Calmodulin-like Protein from Entamoeba histolytica: Solution Structure and Calcium-Binding Properties of a Partially Folded Protein

Calmodulin-like Protein from Entamoeba histolytica: Solution Structure and Calcium-Binding Properties of a Partially Folded Protein

Root‐mean‐square‐deviation‐based rapid backbone resonance assignments in proteins

Spectroscopic labeling of A, S/T in the 1H–15N HSQC spectrum of uniformly (15N–13C) labeled proteins

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