Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.

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Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.

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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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09-13-2011, 08:27 PM

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Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.

Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.

Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies.

Protein Expr Purif. 2011 Aug 31;

Authors: Chen W, Gamache E, Richardson D, Du Z, Wang C

Abstract
Alzheimer's disease (AD) is the most common type of dementia in elderly people. Senile plaques, a pathologic hallmark of AD, are composed of amyloid ? peptide (A?). A? aggregation produces toxic oligomers and fibrils, causing neuronal dysfunction and memory loss. A? is generated from two sequential proteolytic cleavages of a membrane protein, amyloid precursor protein (APP), by ?- and ?-secretases. The transmembrane (TM) domain of APP, APPTM, is the substrate of ?-secretase for A? production. The interaction between APPTM and ?-secretase determines the production of different species of A?. Although numerous experimental and theoretical studies of APPTM structure exist, experimental 3D structure of APPTM has not been obtained at atomic resolution. Using the pETM41 vector, we successfully expressed an MBP-APPTM fusion protein. By combining Ni-NTA chromatography, TEV protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically-labeled APPTM for NMR studies. The reconstitution of APPTM into micelles yielded high quality 2D (15)N-(1)H HSQC spectra. This reliable method for APPTM expression and purification lays a good foundation for future structural studies of APPTM using NMR.

PMID: 21907289 [PubMed - as supplied by publisher]

Source: PubMed

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