NMR paper Expression in Pichia pastoris and characterization by circular dichroism and NMR of r

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[NMR paper] Expression in Pichia pastoris and characterization by circular dichroism and NMR of r

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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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11-19-2010, 08:32 PM

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Expression in Pichia pastoris and characterization by circular dichroism and NMR of r

Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin.

Related Articles Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin.

Proteins. 2001 Jun 1;43(4):499-508

Authors: Guo RT, Chou LJ, Chen YC, Chen CY, Pari K, Jen CJ, Lo SJ, Huang SL, Lee CY, Chang TW, Chaung WJ

Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma. Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin alpha(IIb)beta3 of platelets. Rho produced in Escherichia coli inhibited platelet aggregation with a K(I) value of 263 nM. Although functional, Rho produced in E. coli is misfolded based on our 2D and 3D NMR studies. In order to correct the folding problem, Rho was expressed in Pichia pastoris. The recombinant Rho expressed in P. pastoris inhibited platelet aggregation with a resulting K(I) value of 70 nM. This is the same potency as that of native Rho. CD analysis showed that the secondary structures of Rho are pH-independent and contain 3.5-7.9% alpha-helix, 48.2-50.5% beta-structures, and 42.3-47% coil. The sequential assignment and structure analysis of Rho were obtained using 2D and 3D 15N-edited NMR spectra. These results provide the first direct evidence that highly disulfide-bonded disintegrin can be expressed in P. pastoris with the correct fold. This evidence may serve as the basis for exploring the structure and function relationships as well as the dynamics of disintegrin and its variants.

PMID: 11340665 [PubMed - indexed for MEDLINE]

Source: PubMed

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