The STINT-NMR Method for Studying In-cell Protein-Protein Interactions.

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The STINT-NMR Method for Studying In-cell Protein-Protein Interactions.

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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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09-05-2010, 05:53 AM

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The STINT-NMR Method for Studying In-cell Protein-Protein Interactions.

The STINT-NMR Method for Studying In-cell Protein-Protein Interactions.

Related Articles The STINT-NMR Method for Studying In-cell Protein-Protein Interactions.

Curr Protoc Protein Sci. 2010 Aug;Chapter 17:Unit17.11

Authors: Burz DS, Shekhtman A

This unit describes critical components and considerations required to study protein-protein structural interactions inside a living cell by using NMR spectroscopy (STINT-NMR). STINT-NMR entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting spectra provide a complete titration of the interaction and define structural details of the interacting surfaces at the level of single amino acid residues. The advantages and limitations of STINT-NMR are discussed, along with the differences between studying macromolecular interactions in vitro and in vivo (in-cell). Also described are considerations in the design of STINT-NMR experiments, focusing on selecting appropriate overexpression plasmid vectors, sample requirements and instrumentation, and the analysis of STINT-NMR data, with specific examples drawn from published works. Applications of STINT-NMR, including an in-cell methodology to post-translationally modify interactor proteins and an in-cell NMR assay for screening small molecule interactor libraries (SMILI-NMR) are presented. Curr. Protoc. Protein Sci. 61:17.11.1-17.11.15. (c) 2010 by John Wiley & Sons, Inc.

PMID: 20814930 [PubMed - in process]

Source: PubMed

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