Biomedical Engineering Reference
In-Depth Information
and NMR spectroscopy.
19
Of these, solution NMR is the most powerful,
providing atomic resolution probes, which in favorable cases are located
uniformly throughout the entire protein of interest. NMR relaxation
dispersion experiments are particularly useful for studying ms-ms motions,
capable of providing kinetic, thermodynamic, and structural information
about the conformational exchange process.
19
The utility of relaxation
dispersion experiments has been demonstrated for a number of proteins and
for a variety of motional processes.
20
Relaxation dispersion experiments have
also been developed for a wide variety of protein nuclei.
21-31
Therefore, it is
not possible to review the methodology and application of relaxation
dispersion in its entirety here. Instead, this chapter will focus on the
application of relaxation dispersion experiments to large enzymes with specific
examples drawn from studies in our lab, while highlighting notable advances
from other research groups.
Large macromolecules have traditionally been problematic to study by NMR
spectroscopy due to spectral crowding and signal-to-noise (S/N) limitations.
However, recently developed NMR pulse sequences and isotopic labelling
schemes have made NMR study of large proteins tractable. These advances have
allowed for the application of relaxation dispersion techniques to systems not
previously amenable to such interrogation. Below, we briefly review the general
theory of relaxation dispersion experiments and discuss their applications to
enzymes with molecular weights larger than 50 kDa.
7.2 Conformational Exchange
Before discussing the application of NMR relaxation dispersion to large
enzymes, we will first briefly familiarise the reader with the idea of
conformational exchange and the basic experimental approaches utilised to
measure relaxation dispersion.
32
A complete treatment of these topics is not
possible in this chapter, so the following discussion will be limited to the case
of two-site conformational exchange of isolated two-spin systems. Discussion
of other exchange systems can be found elsewhere.
33,34
A two-site conforma-
tional
exchange
between
distinct
magnetic
environments,
A
and
B,
is
represented by the following equation,
k
1
A
/
?
B
ð
7
:
1
Þ
k
{1
where the overall rate constant for conformational exchange, k
ex
, is the sum of
the rate constants k
1
and k
21
. This motion is classified as fast, slow, or
intermediate if k
ex
is greater than, less than, or approximately equal to,
respectively, the chemical shift difference, Dv, between a nucleus in
conformation A and that same nucleus in conformation B. Stochastic
fluctuation between these two conformations modulates the resonance
frequency of the exchanging nucleus, increases the transverse relaxation rate,
