- The sample is gradient-shimmed, optimally using a round of
three-dimensional shimming and then one-dimensional shim-
ming until no further improvement. - The^1 H90pulse is experimentally optimized (pulse program
zg, with O1 offset from the water) for individual samples by
observing a 360null. - The^1 H90pulse is copied forward into experiments with the
ZZ-exchange pulse sequence (hsqcexf3gpwgph). Normally the
(^15) N pulse does not need optimization, and the default pulse
is used.
- A series of 2D experiments are separately set up and acquired
sequentially with a varying mixing time delay (d7) for exchange.
For example, for here delays of 0.03, 0.05, 0.09, 0.13, 0.15,
0.35, 0.55, 0.75, and 0.95 s are used and collected in random
order. The experiments are then performed with 2048t 2 and
128 t 1 points with 64 scans pert 1 point (seeNote 4). - Spectra are processed using NMRPipe [16] or similar software
(seeNote 5). - For resolved resonances, peak volumes are measured with
SPARKY (DG Kneller, I. K. UCSF Sparky-an NMR display,
annotation and assignment tool.University of California, San
Francisco,(1993)) or similar software. - Peak volumes could then be analyzed with MATLAB scripts
provided by Dr. Demers and Dr. Mittermaier [5]. Values ofkoff
are obtained by a global fit of all the data andkonvalues
determined with the use of ITC-derivedKdvalues. The initial
round of data fitting occurs on a per residue basis. At this stage
each residue will have its own plot and fit allowing the user to
identify residues with weak data or regions of overlapping
intensities. These “poor residues” can then be excluded from
further analysis. In the final step, a global analysis is performed
where all residues are fitted together and Monte Carlo simula-
tions are performed to obtainkoffand NMR derivedkonvalues
as well as their errors (seeNote 6).
3.4 CPMG Relaxation
Dispersion NMR
Spectroscopy
Measurements
- A 500 μM sample of β1-CBM is mixed with a
sub-stoichiometric amount (20 μM) of carbohydrate. For
other CBMs and carbohydrates, the optimal amount requires
trial and error until a suitable dispersion signal is obtained. - The sample is transferred into a 5 mm NMR tube and included
5%^2 H 2 O for locking. - The pulse sequence for CPMG relaxation dispersion used on the
Bruker spectrometer is encoded in the library as hsqcrexetf3gp-
si3d as a pseudo 3D experiment with single scan interleaving.
Carbohydrate Binding Kinetics of theβ-Subunit CBM 95