Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations.

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Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations.

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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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09-30-2011, 05:59 AM

nmrlearner

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Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations.

Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations.

Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations.

J Biomol NMR. 2011 Sep;51(1-2):5-19

Authors: McIntosh LP, Naito D, Baturin SJ, Okon M, Joshi MD, Nielsen JE

Abstract
NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK (A) values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain (13)C(?) nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK (A) values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK (Ai) values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK (A) values and hence catalytic roles of these two residues result from their electrostatic coupling.

PMID: 21947911 [PubMed - in process]

Source: PubMed

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