AMPK Methods and Protocols

C2-bound AMPK to co-crystallize [10]. (2) Structural analysis of

the α1-RIM reveals it forms a stronger interaction with the

γ-subunit than theα2-RIM; therefore, we utilized aα1/2-RIM

swap chimera for crystallization studies [10]. We have optimized

conditions for the two crystallization constructs based on the pre-

viously published method by Xiao and colleagues [6].

1.3 Cryoprotection
of AMPK Crystals

Cryoprotection is the most important step in producing high-

quality diffracting AMPK crystals but also the most variable. Cryo-

protectants are used to protect the protein crystals against freezing

damage by ice formation during flash cooling. Xiao and colleagues

reported that the reservoir solution supplemented with 30% ethyl-

ene glycol sufficiently protects AMPK crystals during flash cooling

[6]; however, in our hands we find the diffractive quality of crystals

cooled in this way differs greatly between samples. We prefer to use

a combination of cryoprotectants mixed in with the reservoir solu-

tion in a stepwise manner.

X-ray crystallographic studies of full-length AMPK are valuable

for the development of therapeutic activators and inhibitors for

AMPK, whether they bind to the ADaM site,α-kinase domain, or

γ-CBS motifs. Here we describe detailed, optimized protocols from

our laboratory for the crystallization of full-lengthα 2 β 1 γ1 AMPK,

including protein production and purification and cryoprotection

of the crystals. The techniques described will allow for visualization

of compounds targeted at all of the known AMPK allosteric sites.

2 Materials

2.1 Expression
and Purification
of AMPK
for Crystallization

1. AMPKα 2 β 1 γ1 dual expression constructs: cDNAs forα2/γ 1
   cloned into pETDuet™-1 multiple cloning sites (MCS)
   1 (BamH1/Not1) and 2 (MfeI/XhoI), respectively, andβ 1
   cDNA cloned into pRSFDuet™-1 MCS1 (NcoI/BamHI).


2. AMPK α2/α1 RIM chimera dual expression constructs:
   cDNAs forα2/1 (α2(1–347)/α1(349–401)/α2(397-end))/
   γ1 cloned into pETDuet™-1 multiple cloning sites (MCS)
   1 (BamHI/Not1) and 2 (MfeI/XhoI), respectively, and
   cDNA forβ1 into pCOLADuet™-1 MCS1 (NcoI/BamHI)
   (seeNote 2).


3. LB (lysogeny broth): 10 g/L tryptone, 5 g/L yeast extract,
   and 10 g/L NaCl.


4. LB agar: 1.5% (w/v) agar in LB media and then autoclave.


5. Ampicillin 1000
   stock: 50 mg/ml ampicillin in water.


6. Kanamycin 1000
   stock: 50 mg/ml kanamycin in water.


7. Autoclaved glycerol.


8. Expression flask: 2 L baffled Erlenmeyer flask.

18 Christopher G. Langendorf et al.