The combination of ITC and NMR is a proven method and has
been used previously for studying protein interactions when con-
ventional methods fail [5, 13]. However, care needs to be taken to
appropriately apply the experiments.β1- andβ2-CBM have differ-
ences in protein motion [14]. When studied by CPMG relaxation
dispersion,β1-CBM appears to have noμs-ms motion in the free or
bound state, whereasβ2-CBM has significant dynamics in both
states. In order to study carbohydrate-binding kinetics of β1-
CBM, CPMG relaxation dispersion experiments, in the presence
of sub-stoichiometric amounts of carbohydrate, were applied, thus
monitoringμs-ms motion. On the other hand, this method could
not be used forβ2-CBM as microsecond motion observed in the
free state could confound results obtained in the presence of
sub-stoichiometric amounts of ligand. As carbohydrate binding to
β2-CBM appears to result in slow exchange, ZZ-exchange spec-
troscopy was used to study the dynamics ofβ2-CBM binding
carbohydrate. These NMR methods were used to obtain dissocia-
tion rates (koff) of carbohydrate binding which could be used in
conjunction with ITC-derived affinity values (Kd) to obtain associ-
ation rates (kon)[ 3].2 Materials
- Purifiedβ1 andβ2 CBMs, Q75 to K156 (usingβ2 numbering)
of the β-subunit of Rattus norvegicus AMPK cloned into
pGEX-6P-3 (seeNote 1).
2.β-cyclodextrin (BCD) (Sigma Aldrich), glucosyl-β-cyclodextrin
(gBCD) (Wako Pure Chemical Industries Ltd.). - Sodium phosphate buffer: 100 mM Na 2 HPO 4 /NaH 2 PO 4 ,
pH 6.8, degassed (seeNote 2). - Mini dialysis kit, 1 kDa cutoff.
- Calorimeter: e.g., MicroCal iTC200 (Malvern).
- NMR spectrometers of two or more field strengths, e.g., Bru-
ker 600–800 MHz of the Avance II or more recent models,
equipped with triple resonance cryoprobes (^1 H,^13 C,^15 N)
(TXI or TCI). - Spectrophotometer: e.g., Bio-Rad Smart Spec 3000 or similar
device. - Hamilton syringe 200μl.
Carbohydrate Binding Kinetics of theβ-Subunit CBM 93