NMR paper Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS.

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[NMR paper] Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS.

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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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08-28-2020, 02:27 PM

nmrlearner

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Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS.

Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS.

Related Articles Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS.

Cell Rep. 2020 Aug 25;32(8):108074

Authors: Zhao Q, Fujimiya R, Kubo S, Marshall CB, Ikura M, Shimada I, Nishida N

Abstract
The small guanosine triphosphatase (GTPase) RAS serves as a molecular switch in signal transduction, and its mutation and aberrant activation are implicated in tumorigenesis. Here, we perform real-time, in-cell nuclear magnetic resonance (NMR) analyses of non-farnesylated RAS to measure time courses of the fraction of the active GTP-bound form (fGTP) within cytosol of live mammalian cells. The observed intracellular fGTP is significantly lower than that measured in*vitro for wild-type RAS as well as oncogenic mutants, due to both decrease of the guanosine diphosphate (GDP)-GTP exchange rate (kex) and increase of GTP hydrolysis rate (khy). In*vitro reconstitution experiments show that highly viscous environments promote a reduction of kex, whereas the increase of khy is stimulated by unidentified cytosolic proteins. This study demonstrates the power of in-cell NMR to directly detect the GTP-bound levels of RAS in mammalian cells, thereby revealing that the khy and kex of RAS are modulated by various intracellular factors.

PMID: 32846131 [PubMed - in process]

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