- As for the ZZ-exchange experiment, the sample is tuned and
shimmed, and the^1 H90pulse is determined. - A list of pulse frequencies (vdlist) is prepared (0, 50, 2x75,
100, 2x150, 200, 300, 500, 2x700, 800, 900, 1000,
1500 Hz). The list is prepared with the frequencies in a random
manner. It is important to setTD1andNBLto the number of
entries in the vdlist (here 16). The length of mixing time per
CPMG block (d21) is typically set to 40 ms, so thatTCPMGis
80 ms. Individual 2D data sets are acquired with 2048t 2 (^1 H)
and 256t 1 (^15 N) points and 24 scans for eacht 1 point (see
Note 7). - Trial experiments are conducted at a single field varying ligand
concentrations until optimal and significantR 2 obsare observed
(Fig.3)[ 17]. Full data collection is then conducted on the
same sample at two magnetic fields. - Data can be processed using various software packages. We use
NMRPipe to process our data (seeNote 8). - The processed NMR data is read into software such as SPARKY
or similar software and peak intensities (heights) measured. For
specific peaks these intensities are fitted to the model using
software packages kindly provided by Dr. Korzhnev
[12]. From these datakoffis determined andkonfrom ITC-
derivedKdvalues (eqs.8 and 10). CPMG dispersion profiles
are initially fit with all parameters calculated on a per residue
basis. Initial fits are observed to determine if reasonable chemi-
cal exchange occurred and residues could be excluded from
further analysis if poor or no chemical shift is observed. In the
final round residues are fit in a global manner where a singlekoff
value can be obtained. If chemical shifts for the unbound and
saturated states are known, chemical shift differences can be
supplied to calculations (seeNote 9).
4 Notes
- Overexpression of the AMPKβ-CBMs inEscherichia coliis
straightforward. For ITC, protein is expressed inE. coliBL21
(DE3) in rich media (2xYT) to produce milligram quantities of
protein. Protein is purified to homogeneity [3]. As the NMR
experiments described here require^15 N–labelling of the protein,
expression inE. coliBL21(DE3) is more demanding, requiring
minimal media with^15 N–ammonium chloride the sole nitrogen
source and glucose as the sole carbon source. Our favored pro-
tocols for obtaining large milligram quantities for NMR experi-
ments are found in these publications [18, 19]. Over the course
of our work, theβ1- andβ2-CBM^1 H,^13 C,^15 N NMR spectra
96 Paul R. Gooley et al.