Chemistry Reference
In-Depth Information
4.1.3 Exploring New Frontiers Using Methyl Protonated Samples
Although methyl protons have been widely used to assist structure determination of
large protein complexes, their high sensitivity and amenability to TROSY-type
sequences (described in Sect.
4.2
) has provided the foundation for exciting
applications that probe large systems in dynamic equilibrium [
256
,
257
]. This
was spectacularly demonstrated for the 670-kDa 20S core-particle proteasome,
with the identification of two interchanging structural states for gating residues
using
13
CH
3
-labeled Met in U-
2
H,
12
C-labeled samples [
271
], along with
interactions inside the proteasome antechamber that maintained ILV-labeled sub-
strate proteins in an unfolded state [
272
]. Meanwhile, applications with membrane
protein samples are also beginning to appear, as shown with the DDM-solubilized
KcsA channel [
273
]. In this study, methyl TROSY spectra from U-
2
H, Leu/Val-
[
13
CH
3
,
12
CD
3
], Tyr-
1
H KcsA showed pH-dependent chemical shifts reflecting
interconversion between three different functional states, and coupling between
gating residues in two distinct regions of the structure (Fig.
4
). Selectively ILV-
labeled samples have also been used to characterize the spectroscopically-invisible
vesicle-bound states of
-synuclein by transfer-NOE type experiments involving
Leu methyl protons that were detected in the
1
H-
15
N HSQC spectrum of the free
state [
274
].
In an even more ambitious application, methyl group signals allowed ligand-
induced changes in the extracellular surface of the 365-residue human
a
2 adrenergic
receptor in DDM to be investigated without the assistance of deuterium labeling
[
275
]. In this study
13
C-methyl groups were introduced through reductive methyla-
tion of solvent-exposed Lys
b
-NH
2
groups. The presence of a salt-bridge interaction
on the extracellular side could be confirmed, with changes in this interaction being
detected in the
1
H-
13
C HMQC spectrum upon agonist binding. This data provided
new insights into the structure of the active state, a form that had not yet been
captured by X-ray crystallography. The relatively large signal-to-noise ratio
provided by methyl groups at the ends of the long flexible Lys side chain was
instrumental for the success of this experiment, particularly given the low
concentrations of samples that were available (60-200
e
m
M). These experiments
illustrate the great potential for methyl protons to provide a window into previously
inaccessible dynamic states of large membrane proteins.
4.2 Relaxation Optimization
Given the large size of typical membrane protein samples, selective labeling
approaches must usually be accompanied by relaxation-optimized NMR
experiments. Just over a decade ago, rapid transverse relaxation rates associated
with large, slowly tumbling molecules defined a ~30-40 kDa molecular weight
ceiling for protein NMR. Beyond this limit, broad peaks and magnetization losses
during coherence transfer thwarted attempts to acquire standard spectra required for
