Biomedical Engineering Reference
In-Depth Information
method increases the precision of the PRE data, but the sensitivity is greatly
reduced, mainly due to the low intensities in the spectra of the second time-
point. In our hands, both methods yield similar results.
6.3.2
Applications for Studying Lowly Populated States
6.3.2.1 Protein-DNA/RNA Interactions
Traditionally, the use of PRE was limited by the availability of naturally
occurring paramagnetic centers. In 2004, Iwahara et al. used a Mn
2+
chelating
tag to study the complex between DNA and the DNA binding sex-determining
region Y (SRY) protein. They attached the tag to a thymine base (dT-EDTA-
Mn
2+
) at either end of a DNA duplex and showed that intermolecular PRE
could be used directly for structure refinement, provided that the dynamics of
the tag were accounted for during data analysis.
36
Later in 2004, Iwahara et al.
also used this approach to study the non-specific binding of the high-mobility
group box-1A (HMGB-1A) to DNA. Unexpected PRE profiles were found at
high salt concentrations leading to the discovery that PRE could be used to
visualise transient, lowly populated states. The PRE profiles suggested that
HMGB-1A moves along the DNA strand as well as hopping from one DNA
molecule to another.
82
In 2006, Iwahara and Clore used PRE to further study the complex process
of target searching by DNA transcription factors. Binding of the transcription
factor HOXD9 was studied using DNA duplexes labelled with dT-EDTA-
Mn
2+
that either did or did not contain the target sequence. Two experiments
were performed on samples containing equal amounts of the DNA duplexes:
one with the specific DNA duplexes labelled, where only searching via
intermolecular translocation could be detected, and one with the non-specific
DNA duplexes labelled, where searching via either inter- or intramolecular
translocation could be detected. It was found that non-specific association
aided in specific complex formation via both inter- and intramolecular
translocation. Transient intermediates were also shown to form at non-specific
sites with similar structures to the specific complex. These results demonstrated
that
PRE
can
provide
valuable
information
on
molecular
recognition
processes as well as structural data for transient intermediates.
83
The data from PRE and RDC measurements can also be combined; RDC
data provide bond vector orientations while PRE data provide highly sensitive
distance restraints as well as the additional data set required to solve the
degenerate orientation of the x-tensor.
4
This has been done for several
unfolded and disordered proteins,
52,60-62
mentioned in Section 6.3.2.4, as well
as for the ternary complex between RNA and the RNA recognition motifs of
proteins Hrp1 and Rna15. In 2010, Leeper et al. recorded RDC and PRE data
for the complex using anchoring RNA (GGAUAUAUAUAAUAAU) with
nitroxide spin-labels at U3 and U5. The structure was determined despite the
large size of the complex, 34 kDa, and the weak interactions between the
