required for the complete HDX-MS profile analysis of AMPK
(42 total injections for a single pairwise comparison for Apo
AMPK vs. Ligand one encompassing seven time points and
three replicates).- Manually inspect data from each peptide to ensure accurate
analysis parameters are used in deuterium level measurements
(e.g., retention time, S/N intensity, and absence of interfering
ion signals from overlapping peptides in m/z bars). - To identify significant differences between individual peptides
across the two protein states (Apo AMPK vs. AMPK-AMP
ligand complex), statistical analysis based on p-values
(p<0.05 for any two time points or<0.01 for a single time
point) from a simple Student’s t test between the replicates
from the bound and free states is used to differentiate true
statistically significant differences in perturbation from experi-
mental noise distribution. - Present the HDX-MS data in an user-desired format such as
time-dependent deuterium uptake plots; sequence coverage
differential perturbation heat map view, overlaid on a 3D
structure or model using PyMOL; experiment comparison
view comparing multiple ligands in a table format; and
residue-level perturbation data extrapolated from overlapping
peptides or a Bayesian approach [11, 32](see Note 31)
(Fig.2).
3.3 X-Ray
Crystallography
Followsteps 1– 5 for crystallization of AMPK.- Design a construct suitable for crystallographic studies of
AMPK [9](seeNote 32). - After purification of recombinant AMPKxtal(seesteps 2– 14 in
Subheading3.1), confirm protein purity by mass spectrometry
and SDS-PAGE analysis. Pool peak fractions and concentrate
to 10–15 mg/ml. Aliquot into 30μl single-use tubes and flash
freeze in liquid nitrogen. Store at 80 C until use. - Prepare a 96-well crystallization grid based on the following
condition: 100 mM trisodium citrate, 750 mM ammonium
sulfate, 500 mM lithium sulfate, and 1% (v/v) ethylene glycol.
Vary concentration of ammonium sulfate and lithium sulfate
systematically (from ~250 mM to ~1 M), and screen for wells
that give largest/best crystals (seeNote 33). If dispensing
directly into 96-well trays (Intelliplates), prepare 70–75μlof
each condition. If mixing manually, prepare 500–1000μlof
each condition and dispense into 24-well plates. - Thaw an aliquot of AMPK on ice. Then add 400μM of staur-
osporine and 400μM of AMP. Incubate on ice ~10 min.
Biophysical Studies to Evaluate Protein-Ligand Interactions 41