3 Methods
3.1 Sample
Preparation for ITC
and NMR
Spectroscopy
- Before ITC experiments, unlabelledβ1- andβ2-CBM protein
samples are dialyzed overnight in 100 mM sodium phosphate
buffer (pH 6.8). Similarly before NMR experiments,^15 N–la-
belledβ1- andβ2-CBM protein samples are dialyzed in the
same buffer system. In all cases, the dialysate is used to prepare
solutions of glucosyl-β-cyclodextrin (gBCD) and
β-cyclodextrin (BCD) of known concentrations. - Protein concentration measurements are based on the absor-
bance at 280 nm. The theoretical extinction coefficient for the
β-CBMs was calculated by ProtParam (http://www.expasy.ch/
tools/protparam.html) based on [15].
3.2 Isothermal
Titration Calorimetry
Measurements
- A 200μl sample of 30μM protein is loaded into the sample cell
using a Hamilton syringe; extra care must be taken to avoid
introducing air bubbles. Samples can be degassed if needed. - More than 40μl of 300μM ligand can be added to a PCR tube
and loaded into the syringe following the instructions from the
ITC software (seeNote 3). - With stirring a titration is performed with 16 injections of
2.5μl of carbohydrate with the sample cell set to a desired
temperature. Each injection is done over a 5 s period with a
180 s delay between injections. - A blank sample of injecting carbohydrate into buffer is per-
formed identically and subtracted from each result so that at
saturation the binding curves should approach zero kcal.mol^1. - The data is fitted using the Origin MicroCal software assuming
a single-site binding model. However, the values for stoichi-
ometry are also fitted, along with the binding affinity (Ka¼1/
Kd) and enthalpy (ΔH).
3.3 ZZ-Exchange
NMR Spectroscopy
Measurements
- A 500μM sample of^15 N–labelledβ2-CBM is mixed with a
sub-stoichiometric amount (250μM) of carbohydrate. The
binding of carbohydrate must fit to a slow exchange condition
such that two signals are observed in the 2D^1 H,^15 N HSQC
(heteronuclear single quantum correlated) spectrum, one
reflecting the free state and another the bound state. This
may require trial and error until the signals are approximately
equal. Also to see exchange the resonances of the free and
bound are ideally resolved in both the^1 H and^15 N dimensions
of a 2D^1 H,^15 N HSQC spectrum (Fig.2). - Once placed in the spectrometer, samples are equilibrated at
25 C or the desired temperature, and^1 H and^15 N channels are
optimally tuned.
94 Paul R. Gooley et al.