Fractional enrichment of proteins using [2- 13 C]-glycerol as the carbon source facilitates measurement of excited state 13 CÎ± chemical shifts with improved sensitivity

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Fractional enrichment of proteins using [2- 13 C]-glycerol as the carbon source facilitates measurement of excited state 13 CÎ± chemical shifts with improved sensitivity

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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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05-20-2015, 10:27 AM

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Fractional enrichment of proteins using [2- 13 C]-glycerol as the carbon source facilitates measurement of excited state 13 CÎ± chemical shifts with improved sensitivity

Fractional enrichment of proteins using [2- 13 C]-glycerol as the carbon source facilitates measurement of excited state 13 CÎ± chemical shifts with improved sensitivity

Abstract

A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13CÎ± chemical shifts using Carrâ??Purcellâ??Meiboomâ??Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the CÎ± positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13CÎ± chemical shifts can be obtained from RD experiments, with errors on the order of 0.06Â*ppm for exchange rates ranging from 100 to 1000Â*sâ??1, despite the small fraction of 13CÎ±â??13CÎ² spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13CÎ± spin probes.

Source: Journal of Biomolecular NMR

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