Chemistry Reference
In-Depth Information
solubilized OmpX. The small quantities involved made it possible to evaluate the
NMR spectral properties of refolded samples in a series of novel detergents that
were available in a very limited supply. Meanwhile, even faster screening can be
achieved if the first set of conditions is evaluated using 1D experiments, with 2D
spectra only being acquired for the most promising samples [
147
]. Also exciting is
the recent availability of the cryogenically cooled 1.7-mm microprobe, with an
increase in mass sensitivity that compensates for the tenfold reduction in sample
volume (30
L in a conventional 5-mm probe). With spectral acquisition
times and sample concentration requirements being comparable to those of the
5-mm room temperature probe, it is in fact now possible to use these reduced
sample volumes for acquisition of all NMR data types required [
147
,
232
].
One aspect of detergent screening that is gaining increasing attention is the
concentration of the detergent itself. While it has long been recognized that there is
a minimum concentration required for solubilization, samples that exceed this
minimum can also exhibit poor spectral quality [
232
]. Screening experiments
with a 1.7-mm microprobe showed that the degradation in OmpX spectral quality
associated with higher decylphoshocholine concentrations was caused by an
increase in the solution viscosity due to the presence of excess protein-free
micelles. Based on these results it could be shown that the optimal detergent
concentration for this system approximately corresponded to the number of deter-
gent molecules bound in the protein-detergent complex multiplied by the concen-
tration of OmpX, added to the cmc of the detergent. High detergent-to-protein ratios
can also complicate the study of oligomeric states of membrane proteins, with
excess micelles reducing the effective concentration of TM segments that can
interact [
243
-
245
]. However, the practice of concentrating samples in ultracentri-
fugation devices can increase the concentration of detergent in the final sample,
even when the molecular weight cut-off is smaller than the size of the protein-free
micelle [
234
,
246
]. This can be particularly problematic for detergents that tend
toward greater polydispersity and concentration dependence of micelle size. Con-
sequently, it is important to choose ultrafiltration units with molecular weight cut-
offs that can maximize retention of the target protein while minimizing undesired
increases in detergent concentrations, particularly for any samples that require
concentration or detergent exchange as a final preparation step. It is useful to
monitor detergent concentrations during these procedures, with detergent peak
intensities in 1D
1
H NMR spectra providing a convenient read-out of concentration
for many of the commonly used detergents for solution NMR [
234
].
m
L vs 300
m
4 Methods for Acquisition of Solution NMR Spectra
of Membrane Proteins
As discussed above, most of the systems used to reconstitute a membrane protein
sample for solution NMR create large, slowly tumbling complexes that suffer
from rapid transverse relaxation processes. Size estimations of some of these
