An investigation of Rheo-NMR techniques to improve the capture of residual dipolar couplings : a thesis submitted in partial fulfillment of the requirements for the award of the degree of Master of Science in Chemistry, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
Residual Dipolar Couplings (RDCs) are an increasingly important structural restraint
that can be used to help generate high quality structural models of proteins
by Nuclear Magnetic Resonance (NMR) methods. They are captured with the
aid of an alignment medium that imposes some anisotropy to the protein’s tumbling.
Current methods for the capture of multiple sets of these couplings are
tedious, expensive, and do not always result in unique sets being captured. This
thesis set out to investigate whether multiple RDC sets could be captured from a
single sample by controllably shearing the liquid crystal alignment medium used.
Initial experiments focused on the ability to controllably realign a number of
different nematic phase liquid crystals. These experiments found that controlling
the director angle of the liquid crystal is possible, and that a number of stable
alignments can be achieved through the application of different shear stresses.
The application of RDCs to small molecules is a very young field that is still
developing and finding potential uses. In this thesis a small molecule system
of (+)-isopinocampheol ((+)-IPC) was investigated with RDCs being collected
from this molecule within a liquid crystal phase with the director at a number of
different orientations relative to the external magnetic field. The fitting of these
captured RDCs to a structural model of the (+)-IPC was not able to generate a
high quality fit for any of the RDC sets collected, leading to some puzzling results.
It is hypothesized that inhomogeneity of the alignment phase was responsible for
As the application of RDCs is so heavily dominated by protein structure studies,
a small protein was investigated. The protein of choice, ubiquitin, has been
heavily investigated in the past, and is often used as a demonstrator protein
for new NMR techniques. This work presents several RDC data sets measured
from ubiquitin which were successfully captured at a variety of different director
orientations of the alignment media. These RDC sets were all successfully fitted
to a previously known X-Ray crystallographic structure of ubiquitin, and unique
alignment tensors for each RDC data set were extracted.
Finally, structure calculations were carried out incorporating these captured
ubiquitin RDC data sets with the goal of investigating how the variation in the
ensembles of structures generated was modified. The results from these calculations
showed that the addition of RDC data (over and above NOE constraints) to
the simulated annealing process results in ensembles of higher quality structures
being obtained. However, the addition of multiple sets of RDC data (collected
with different director alignments) did not appear to cause any further improvement.