Biomedical Engineering Reference
In-Depth Information
structure determination, using X-ray diffraction or NMR spectroscopy,
generally ignores protein motions, and represents the presence of rapidly
exchanging conformational equilibria in terms of a static structure.
NMR is uniquely suited to the simultaneous determination of protein
structure and dynamics in solution, as all NMR spectra report on a
conformational average on timescales up to the millisecond. Each measurable
resonance represents an average of rapidly interchanging conformations whose
difference in chemical shift is smaller than their interconversion rates. The
correct interpretation of this almost overwhelmingly vast conformational
average underpins NMR-based structural biology, and has understandably
attracted a great deal of attention. Unfortunately, despite the high sensitivity
of NMR to protein conformational sampling, not all experimentally measured
parameters can be interpreted in a tractable manner in terms of biomolecular
motions. Progress in the prediction of protein chemical shifts has led to their
use in ab initio structure determination,
5-7
but it is not yet possible to use the
population-weighted chemical shift itself to accurately describe the conforma-
tional dynamics of the protein. Residual dipolar couplings (RDCs), are
exquisitely sensitive to orientational dynamics occurring on essentially the
same timescales as the chemical shift and therefore offer a very promising tool
for probing physiologically important motions in biomolecules.
8-10
NMR is routinely used to probe protein motions using spin relaxation,
which describes the mechanisms which return an excited nuclear spin state to
equilibrium.
15
N and
13
C relaxation rates, commonly measured in isotopically
labelled biological macromolecules in solution, are dominated by the random
re-orientational properties of relaxation-active interactions inducing local
fields in the vicinity of the observed spin.
11
In the case of
15
N relaxation,
relaxation rates measured at static magnetic fields up to 20 T report essentially
on the angular re-orientational correlation function of internuclear bond
vectors. Spin relaxation is sensitive to motions on timescales that are faster
than the characteristic molecular rotational diffusion time constant t
c
(in the
range of 5-30 nanoseconds for typical soluble proteins), and these measure-
ments are commonly made for nuclear spin pairs distributed throughout the
protein, and interpreted in terms of amplitudes and frequencies of local
structural fluctuations. Dynamics occurring on timescales in the nano- to
micro-, and even millisecond range are potentially of even greater interest,
because many biologically important processes, such as enzymatic catalysis,
signal transduction, ligand binding and allosteric regulation are expected to
occur on these timescales. Consequently, over the last decade there has been
substantial
development
of
techniques
to
accurately
probe
these
slower
timescale motions at atomic resolution using RDCs.
In this chapter, a brief review of the use of RDCs for the simultaneous study
of protein structure and dynamics will be presented, including a description of
recent approaches that provide a quantitative description of the extent and
nature of intrinsic dynamics in folded proteins in solution.
