Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics

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Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics

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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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10-11-2023, 03:50 PM

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Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics

Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics

Abstract

In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel Î±-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13Câ??1Â*H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13C-T2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-Ï? interactions in protein-ligand complexes.

Source: Journal of Biomolecular NMR

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