Modulation of enzymatic activity of proteins by small molecules can
be probed by classical enzymology methods which reveal insights
into effects on the Michaelis-Menten constant (Km) or the maximal
reaction rate (Vmax). Interactions of small molecule synthetic acti-
vators of AMP-activated protein kinase (AMPK) have been probed
by the application of these biochemical and biophysical methods.
AMPK is a heterotrimeric serine/threonine protein kinase that
regulates cellular and whole-body energy homeostasis in response
to metabolic stress. It consists of a catalyticαsubunit, which con-
tains the kinase module, and two regulatory subunits,βandγ.In
vivo, it is regulated by reversible phosphorylation as well as by
binding of nucleotides to its γ subunit [1–4]. Activation of
AMPK by small molecule synthetic activators has been pursued as
a viable strategy for the development of novel therapeutics for
insulin resistance and metabolic syndrome [5–8]. We have probed
the interactions of AMPK with small molecules using a number of
methods including X-ray crystallography, SPR, HDX, and enzy-
matic assays which are briefly described below [9–11]. Also
included is a brief description of two molecular biology methods
that we have used to generate recombinant AMPK protein
reagents.1.1 Recombinant
Protein Production
All biophysical techniques described below rely on the availability of
highly pure and homogenous recombinant AMPK. As expression
of individual subunits (either inE. colior insect cells) did not yield
sufficient quantities of soluble protein (in-house unpublished data),
we pursued simultaneous expression of AMPK subunits. As
reported by Neumann et al., a single plasmid carrying all three
subunits of AMPKα 1 β 1 γ1 driven by T7 RNA polymerase as a
single tricistronic messenger inE. coliallows spontaneous forma-
tion of the heterotrimeric complex in the bacterial cytosol
[12]. Using this method we generated multiple combinations of
full-length and truncated mammalian AMPK complexes inE. coli
[9, 11, 13, 14]. In addition, we have also produced recombinant
AMPK in insect cells by co-transfection with three viruses that
contain individual AMPKα,β,orγsubunits (seeNote 1).1.2 Hydrogen/
Deuterium Exchange
Coupled to Mass
Spectrometry
Hydrogen/deuterium exchange (HDX) coupled to mass spec-
trometry is a powerful technique to investigate protein structure
and conformational dynamics as it reports on changes in solvent
deuterium uptake on the backbone amides in response to chemical
and posttranslational modifications (PTMs), mutations, and ligand
binding events [15, 16]. High-resolution structural tools such as
X-ray crystallography and NMR provide a detailed picture of a
protein’s structure and ligand binding mode. However, crystal
structures can only provide a single snapshot of a protein’s confor-
mation ensemble, and protein dynamics studies using NMR are
often limited to small molecular weight protein systems that are30 Ravi G. Kurumbail et al.