Characterization of Sarcoplasmic Reticulum Ca(2+) ATPase Nucleotide Binding Domain Mutants using NMR spectroscopy.

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Characterization of Sarcoplasmic Reticulum Ca(2+) ATPase Nucleotide Binding Domain Mutants using NMR spectroscopy.

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NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

Preditor

TALOS

Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

PECAN

Flexibility from chemical shifts:

RCI

Interactions from chemical shifts:

HADDOCK

Chemical shifts re-referencing:

NMR model quality:

NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

Quality Control Check

NMR spectrum prediction:

FANDAS

MestReS

V-NMR

Flexibility from structure:

Backbone S2

Methyl S2

B-factor

Molecular dynamics:

Gromacs

Amber

Antechamber

Chemical shifts prediction:

From structure:

Shiftx2

Sparta+

Camshift

CH3shift- Methyl

ArShift- Aromatic

ShiftS

Proshift

PPM

CheShift-2- Cα

From sequence:

Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

CCPN

From NMR-STAR 3.1

Validate NMR-STAR 3.1

NMR sample preparation:

Protein disorder:

DisMeta

Protein solubility:

Isotope labeling:

Solid-state NMR:

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12-29-2010, 04:04 PM

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Characterization of Sarcoplasmic Reticulum Ca(2+) ATPase Nucleotide Binding Domain Mutants using NMR spectroscopy.

Characterization of Sarcoplasmic Reticulum Ca(2+) ATPase Nucleotide Binding Domain Mutants using NMR spectroscopy.

Characterization of Sarcoplasmic Reticulum Ca(2+) ATPase Nucleotide Binding Domain Mutants using NMR spectroscopy.

Biochem Biophys Res Commun. 2010 Dec 24;

Authors: Myint W, Gong Q, Ahn J, Ishima R

Sarcoplasmic reticulum Ca(2+) ATPase (SERCA) is essential for muscle function by transporting Ca(2+) from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolated SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. (15)N-(1)H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP-PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier's Disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP-PNP dissociation constant (~2.5 mM) was similar to that of WT (~3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.

PMID: 21187073 [PubMed - as supplied by publisher]

Source: PubMed

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