Biomedical Engineering Reference
In-Depth Information
protein-protein interfaces with asymmetric methyl-labelling patterns.
55
The
high sensitivity of the methyl group signal has also been exploited for screening
ligands using NMR spectroscopy.
38
In addition to intermolecular contacts,
methyl-specific protonation of highly deuterated proteins has also permitted
the detection of weak intramolecular interactions.
56
Specific methyl group
protonation in perdeuterated proteins not only reduces the complexity of
Nuclear Overhauser Effect Spectroscopy (NOESY) data, it also considerably
reduces spin diffusion and allows the detection of ultra-long range NOEs.
57
NOEs involving specifically protonated methyl groups have also been used to
aid molecular docking calculations.
58
Stereospecific labelling of methyl groups
using acetolactate can aid methyl group resonance assignment and therefore
the precision of structure calculations of small proteins.
59
More widespread application of methyl-labelling NMR techniques for
studying supramolecular protein systems awaits improvements in resonance-
assignment strategies. Structural and dynamic information yielded by methyl
groups is most useful when a sequence-specific assignment for the probe is
known. Dividing multimeric or large proteins into smaller, more tractable
pieces is not always possible. Likewise, assignment-by-mutagenesis, even in its
most streamlined form, may not be always feasible. Obtaining sequence-
specific resonance assignments of methyl resonances remains the major
bottleneck in many studies of large proteins and protein assemblies by NMR
spectroscopy. The future development of more efficient, user-friendly and
general approaches for resonance assignment will undoubtedly help to extend
the field of application of NMR spectroscopy in this arena.
Continuing progress in isotope-labelling approaches, improvements in
NMR spectroscopy techniques and hardware, and the introduction of widely
applicable assignment strategies, will mean that the use of solution and solid-
state NMR spectroscopy to study high molecular weight proteins becomes
more commonplace. As a result, structural biologists will be able complement
structural data acquired using medium- and high-resolution techniques (e.g.,
X-ray crystallography, small-angle scattering and cryogenic electron micro-
scopy, etc.) with the unique atomic-resolution insight offered by NMR
spectroscopy. Such integrated studies will allow the structural biology of large
proteins and protein assemblies to be addressed at previously unachievable
levels.
Acknowledgements
We thank C. Amero, I. Ayala, E. Crublet, P. Gans, O. Hamelin, R. Kerfah, P.
Macek, M. Noirclerc-Savoye, O. Pessey, R. Sounier and T. Vernet for
stimulating discussions, assistance in sample preparation, and/or critical
reading of this manuscript; the Partnership for Structural Biology for access to
high-field NMR and isotopic-labelling platforms. JB acknowledges funding
from ANR (ANR-09-PIRIBio-445583) and ERC (ERC-Stg-2010-260887), and
