4 Notes
- Levels of AMPK phosphorylation induced by CaMKK2 are
critical for producing protein that will crystallize; high levels
of phosphorylation on theβ1-subunit will inhibit crystalliza-
tion. Analyzing protein phosphorylation using protein TOF
mass spectrometry, we see between one and two individual
phosphorylation events on theβ1-subunit of protein that read-
ily crystallizes. Inhibition of crystallization occurs when there is
between three and six separate phosphorylation events on the
β1-subunit of AMPK. - Once the glycerol stock has been made,steps 1– 6 can be
skipped for future preparations. - We see increased yields of AMPK using the pCOLADuet™-1/
pETDuet™-1 system over the pRSFDuet™-1/pETDuet™-1.
A number of crystallization studies have successfully used tri-
cistronic vectors for AMPK heterotrimer expression [6, 7, 11]. - Check the optical density of the expression culture in a 1 ml
disposable plastic cuvette hourly by spectrophotometry at a
wavelength of 600 nm using LB as a blank. - We see increased yields of AMPK when inducing protein
expression during stationary phase (OD 600 ¼3.0), compared
to inducing during exponential growth phase
(OD 600 ¼0.6–1.0). - The ratio of A-769662 to AMPK is crucial for
co-crystallization, and concentrations of A-769662 above two-
fold of AMPK inhibit crystallization. In addition we have found
that co-crystallization of different ADaM site compounds is
more likely to be successful when their affinity for AMPK is
<500 nM. - The synthetic peptides have not been resolved in the X-ray
crystal structures. Only very weak density has been obtained
for a few residues of the peptide. - The cocamidopropyl betaine concentration we use,
0.0005–0.003%, is quite different to the concentration used
by other researchers, 0.15% [6, 10]. This could be due to
concentration discrepancies between the suppliers. We recom-
mend using a broad concentration range of cocamidopropyl
betaine for initial screening. - Protein feeding experiments involve adding more protein
directly to a crystallization drop containing crystals. Prepare
the protein sample at 4 mg/ml, diluted with storage buffer and
aforementioned compounds.
Visualization of Drug Binding Sites 25