multiple freeze/thaw cycles from the same stock to maintain
protein stability.
- Solubility of AMPK ligands at high concentrations in aqueous
HDX buffers needs to be tested and liganded samples should
be devoid of protein aggregation inducers.
- Columns and connector tubings need to be replaced if the
pressure rises above normal operating ranges. Clogged pepsin
columns can lead to significant peptide carry-over between
experiments.
- Blank injections with buffer (no protein) are included in
between sample injections in the macros for HDX methods
to estimate peptide carry-over between experiments.
- The LC system, but not the protease column, is washed (3)
using a 2-propanol:ACN (2:1 ratio), 0.3% FA gradient
between HDX experiments to ensure that all columns and
lines are clean and free of material from prior injections.
- Repeated injections can likely clog the pepsin column, and
blank HDX runs (a full empty run) with buffer only are
required as a monthly routine procedure to maintain the life-
time of pepsin columns. Never expose the pepsin column to
pH above 8.0 as pepsin will be irreversibly inactivated.
- Sophisticated software and significant computational power is
necessary to handle processing of hundreds of peptides with
multiple replicates over multiple time points.
- While localization of perturbation HDX differences are often
limited to peptide level changes, single amide level resolution
can be achieved using overlapping peptides, a newly devised
Bayesian approach [21], or through alternative fragmentation
techniques such as electron transfer dissociation (ETD) and
electron capture dissociation (ECD) available in many state-of-
the art mass spectrometers.
- To enable crystallographic studies of activators bound to
AMPK at the allosteric drug and metabolite (ADaM) site,
generate a slightly modified form of rat AMPK (called AMPKx-
tal) that contains full-lengthγsubunit but incorporates the
following changes on theαand β subunits [9] A flexible
segment of theα subunit (470–524) is replaced with an
8 amino acid linker (ASGGPGGS) while the first 67 amino
acids of theβsubunit are eliminated. Followsteps 2– 14 in
Subheading3.1 for expression and purification of the crystal-
lographic construct. In the last step of the purification, elute
the AMPKxtalprotein from the sizing column with the crystal-
lographic SEC buffer (seeitem 1in Subheading2.3).
- Once initial crystallization conditions are identified, further
optimization is usually done by systematic screening around
Biophysical Studies to Evaluate Protein-Ligand Interactions 51
