complicated by the need for labeled protein reagents. Protein func-
tion is governed by local and global structural fluctuations that can
be quite complex for large protein complexes such as AMPK het-
erotrimers that contain multiple binding pockets within all three
subunits [17–20]. HDX is a reliable tool to examine such confor-
mational fluctuations. This technique can reveal the nuances of
allosteric communication within and between subunits and provide
insights into mechanism of enzyme activation [11, 16, 21].1.3 X-Ray
Crystallography
X-ray crystallography is the most widely used method to study
three-dimensional structures of proteins. Structure-based drug
design critically depends on insights into the binding modes of
ligands in druggable binding sites on proteins, usually revealed by
crystallography [22]. Well-ordered crystals are produced from
homogenous protein derived from recombinant methods or from
natural sources. Methods such as hanging drop or sitting drop
vapor diffusion are typically used to produce protein crystals
[23, 24]. These are then flash frozen in liquid nitrogen or liquid
ethane in the presence of suitable cryoprotectants prior to X-ray
diffraction experiments. At present, most diffraction measurements
are made at dedicated high-energy synchrotron laboratories around
the world. Sophisticated computational algorithms that employ
Fourier mathematics of the measured X-ray diffraction data reveal
the distribution of electron density at near-atomic resolution.
Ligands are either soaked into preexisting crystals of proteins, or
crystals are generated after forming the protein-ligand complex in
solution in order to gain information on binding of ligands.1.4 Enzymatic
Assays
The rate of catalysis of enzymes varies as a function of substrate
concentration in a hyperbolic fashion. At low substrate concentra-
tion (S), the reaction velocity is a linear function of (S), but it is
independent of (S) at saturating concentrations. By following the
rate of product formation at different substrate and enzyme con-
centrations, we can derive theVmax(the maximum velocity) orKm
(Michaelis-Menten constant, concentration of substrate required
to reach ½Vmax) assuming steady-state kinetics. Enzyme activators
can bring about allosteric changes on proteins through effects on
eitherVmaxorKmor both. As is true for all protein kinases, AMPK
has two substrates: ATP/Mg2+and a peptide or protein substrate.
We have used a 15 amino acid peptide fragment known as SAMS
peptide derived from one of the well-studied AMPK substrates,
ACC1 (acetyl-CoA carboxylase 1), for our biochemical assays and
mechanistic studies [25]. By varying the concentration of one
substrate while keeping the other fixed at tenfold itsKmvalue, we
have determined the effects of small molecule enzyme activators on
the kinetic parameters of AMPK [13, 26].
The biochemical assay method we have employed to monitor
the enzyme kinetics of AMPK is a^33 P-ATP-based filter assay. In ourBiophysical Studies to Evaluate Protein-Ligand Interactions 31