Rheo-NMR studies of macromolecules : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Physics at Massey University, Palmerston North, New Zealand
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Date
2008
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Massey University
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Abstract
In this thesis, the effects of simple shear flow on macromolecular structure and
interactions are investigated in detail via a combination of Nuclear Magnetic Resonance
(NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR
couette shear cell and benchtop shear cell, developed in-house, demonstrated that the
direct measurement of the above phenomena is possible.
First, to determine whether the shear cells were creating simple shear flow, results were
reproduced from literature studies of liquid crystal systems which report shear effects
on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and
Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol.
Next, the possible conformational changes to protein structure brought about by shear
were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein
was sheared, a small, irreversible conformational change was observed by means of
one-dimensional and two-dimensional 1H NMR with reasonable reproducibility.
However, no observable change was detected by means of light scattering. A large
conformational change was observed after shearing a destabilized -Lg sample
containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the
sheared state was similar to the structure of -Lg containing large amounts of -helices
and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils.
Gel electrophoresis tests suggested that the changes were caused by hydrophobic
interactions. Unfortunately, this proved to be difficult to reproduce.
The effect of shear on an inter-macromolecular interaction was investigated by applying
shear during an enzyme reaction of pectin methylesterase (PME) on pectin.
Experimental method and analysis developments are described in detail. It was
observed that under the conditions studied, shear does not interfere with the
de-esterification of pectin with two types of PME, which have different action
mechanisms at average shear rates up to 1570 s-1.
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Keywords
Macromolecular structure, Shear, Pectin, Protein interactions