NMR paper Synthesis of region-labelled proteins for NMR studies by in vitro translation of colu

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[NMR paper] Synthesis of region-labelled proteins for NMR studies by in vitro translation of colu

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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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08-22-2010, 03:03 PM

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Synthesis of region-labelled proteins for NMR studies by in vitro translation of colu

Synthesis of region-labelled proteins for NMR studies by in vitro translation of column-coupled mRNAs.

Related Articles Synthesis of region-labelled proteins for NMR studies by in vitro translation of column-coupled mRNAs.

Biochimie. 1997 Jul;79(7):415-22

Authors: Pavlov MY, Freistroffer DV, Ehrenberg M

A method to synthesise region-labelled proteins for structural studies with NMR is suggested. The technique is based on in vitro translation of matrix-coupled mRNAs. Translation starts with unlabelled amino acids from the initiation codon of the mRNA and continues to the beginning of the region of interest. Here, the ribosomes pause while the tRNAs charged with unlabelled amino acids are replaced with tRNAs charged with isotope-labelled amino acids. Translation then proceeds through the region of interest until the ribosomes pause at its end. At this point aminoacyl-tRNAs are changed again. Translation is resumed with unlabelled amino acids and continues to the STOP codon of the mRNA, where the ribosomes pause. In the final step the complete, region-labelled protein is eluted from the column in almost pure form. The method is demonstrated for small scale synthesis of the DNA binding domain (DBD) of the glucocorticoid receptor (GR), where the DNA-recognising helix is labelled but the rest of DBD is unlabelled. The new technique can be generalised to allow a desired region in a protein to be isotope-labelled.

PMID: 9352091 [PubMed - indexed for MEDLINE]

Source: PubMed

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