Biomedical Engineering Reference
In-Depth Information
oligomerisation
and
aggregation;
thus,
these
studies
could
provide
new
therapeutic targets.
60,104
6.3.2.5 Weak Self-Association
Weak self-association is important for a variety of assembly and maturation
processes.
2
In 2008, Tang et al. showed that PRE could be used to visualise
transient intramolecular interactions for HPr. By recording PRE profiles with
Mn
2+
-chelating tags at three different locations, self-association of HPr was
shown to occur. Based on a concentration of 0.3 mM and a dimer population
in solution of approximately 1%, a K
d
of 15 mM was determined.
105
In 2008,
Tang et al. also used PRE to study the HIV-1 protease precursor, which
undergoes autocleavage of the N-terminal extension during maturation to
form a dimer. PRE data from two spin-labelled precursors with different N-
terminal extensions showed transient association between the N-termini and
the active site. This association enabled autocleavage when the correct
orientation was sampled. The encounter complex was also found to occupy a
wide range of orientations with the conformation of the mature dimer
accounting for only a small fraction. This could explain the low enzymatic
activity of the precursor.
106
Together, these studies demonstrated the use of
PRE for observing weak self-associations that are difficult to visualise with
conventional methods.
6.4
Relaxation Dispersion
6.4.1 RD Theory
Conversion between conformations often involves large motions that expose
nuclei to two or more distinct environments. This is referred to as chemical
exchange. RD aims to quantify the contribution of chemical exchange to the
transverse relaxation rate in order to provide site-specific structural and kinetic
information.
1
RD utilises the difference in resonance frequency in rad/s, Dv, of the active
nuclei
between
the
major
and
the
minor
states
to
probe
several
nuclei
simultaneously eqn (6.8):
Dv~c
I
B
0
(DdzDPCSzDCSA)
ð
6
:
8
Þ
where Dd is the diamagnetic chemical shift difference between the states, DPCS
is the shift difference due to PCS and DCSA is the shift difference due to
chemical shift anisotropy. The Dv between the states can be increased by using
paramagnetic centers to generate PCS, which also provide additional data in
the form of structural restraints.
107
RD requires a system in which the exchange rate, k
ex
, is of a similar order of
magnitude as Dv so that the relaxation rate of the major species is enhanced by
