have been assigned. For any ligand binding study, it is desirable
to assign at least the backbone (^1 HN,^15 N,^13 Cα,^13 Cβ) reso-
nances. Once assigned, titrations of^15 N–labelled protein moni-
tored by^1 H,^15 N HSQC usually reveal the number of binding
sites andKd, complementing ITC experiments. Further these
experiments will inform whether ligand binding fits the slow or
fast exchanging regime. However, to conduct the experiments,
we have described in this article one does not absolutely require
assignments to obtain information from ZZ-exchange or
CPMG relaxation dispersion NMR spectroscopy. Nevertheless,
we note for proteins up to about 15 kDa software and technical
improvements have made assignment of backbone resonances
straightforward and we encourage this approach as considerable
ligand binding and protein behavior information are gained.- As NMR experiments are conducted with relative high concen-
trations of protein and ligand, it is important that buffers are of
a reasonable concentration, as pH may change. We suggest at
least 50 mM and to avoid buffers such as PBS (phosphate
buffer saline). - Appropriate protein and ligand concentrations are determined
by trial and error as the quality of the signal is dependent onKd
andΔH. - Number oft 1 points and spectral widths are experimentally
determined. Also to improve resolution (in F1), isolated
peaks can be folded into the spectrum and the spectral width
in that dimension reduced. - NMRPipe scripts for processing ZZ-exchange are simple
scripts for 2D data and follow the same processing as applied
to a^1 H,^15 N HSQC spectrum. - For estimation of errors of the nonlinear fits of ZZ-exchange
spectroscopy,see[5]. - Number oft 1 points and spectral widths are experimentally
determined. Also to improve resolution (in F1), isolated
peaks can be folded into the spectrum. In deciding the number
of data sets to collect, it is typical to repeat three frequency
points in the CPMG to determine significance of data. - NMRPipe script for separating the pseudo 3D CPMG relaxa-
tion dispersion data into single 2D files. These single files can
then be processed similarly to a 2D^1 H,^15 N HSQC spectrum.
bruk2pipe -in ./ser \
-bad 0.0 -aswap -DMX -decim 2218.66666666667 -dspfvs
20 -grpdly 67.9862518310547 \
-xN 2048 -yN 16 -zN 256 \
-xT 1024 -yT 16 -zT 128 \
-xMODE DQD -yMODE real -zMODE Echo-AntiEcho \
Carbohydrate Binding Kinetics of theβ-Subunit CBM 97