Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.

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$Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.$ Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.

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NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

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GeNMR

I-TASSER

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Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

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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:

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Pseudocontact shifts:

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V-NMR

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Backbone S2

Methyl S2

B-factor

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Shiftx2

Sparta+

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ShiftS

Proshift

PPM

CheShift-2- Cα

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Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

CCPN

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NMR sample preparation:

Protein disorder:

DisMeta

Protein solubility:

Isotope labeling:

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08-25-2010, 02:04 PM

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Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.

Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.

Related Articles Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.

J Biomol NMR. 2010 Aug 24;

Authors: Kitevski-Leblanc JL, Evanics F, Scott Prosser R

In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, (19)F and (1)H-(15)N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of complete (>95%) 3-fluorophenylalanine labeling. The effects of varying levels of enrichment of 3-fluorophenylalanine (i.e. between 3 and >95%) were further studied by (19)F and (1)H-(15)N HSQC spectra,(15)N T(1) and T(2) relaxation measurements, (19)F T(2) relaxation, translational diffusion and heat denaturation experiments via circular dichroism. Our results show that while several properties, including translational diffusion and thermal stability show little variation between non-fluorinated and >95% (19)F labeled samples, (19)F and (1)H-(15)N HSQC spectra show significant improvements in line widths and resolution at or below 76% enrichment. Moreover, high levels of fluorination (>80%) appear to increase protein disorder as evidenced by backbone (15)N dynamics. In this study, reasonable signal to noise can be achieved between 60-76% (19)F enrichment, without any detectable perturbations from labeling.

PMID: 20734112 [PubMed - as supplied by publisher]

Source: PubMed

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