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Biological Chemistry
Nuclear Magnetic Resonance
Dr Max Keniry
http://rsc.anu.edu.au/research/keniry.php
One of the great challenges of
contemporary Nuclear Magnetic Resonance (NMR) spectroscopy is the
application of the technique to highly complex problems in biology.
No other form of spectroscopy can contribute to the elucidation of
the structure, function, and dynamics of biomacromolecules at the
atomic level. Our research is focused on the following three broad
areas: the structure of complexes between DNA and anticancer
antibiotics; the structure of unusual forms of DNA that have
biological significance; and the structure and function of moderately
sized proteins. Work is continuing on structural studies of a large
protein-protein complex, the structures of small chaperone proteins,
and the investigation of the interaction of spermine with various
forms of DNA. As our expertise in macromolecular structure
determinations increases we intend to tackle more demanding
structural problems. In the near future, we will attempt the
structure determination of the N-terminal and C-terminal
domains of a 42 kDa protein that is overexpressed in the cells of
early breast cancer tumours. The ultimate goal of this work is to
use the structure of the protein to design drugs that may be used to
block the progression of the tumour cells. The major theme of our
work is to deduce the function of biological molecules and complexes
from knowledge of their structure and dynamics at the atomic level.
Interaction of the theta Subunit and the e Subunit
of DNA Polymerase III
The
catalytic core of Escherichia coli DNA polymerase III contains
three tightly associated subunits (a, e,
and theta). The refinement of the
three-dimensional structure of the theta
subunit was completed by the NMR group. The theta
subunit has three a-helices in the
N-terminal two thirds of the protein that fold to form a
triangular shape. The surface of theta is
bipolar; with one face containing most of the acidic residues and the
other face most of the long-chain basic residues. The C-terminal
section of theta has many charged and
hydrophilic amino acid residues, but has no well-defined secondary
structure, and exists in a highly dynamic state. This dynamic
conformational exchange in the helices hindered the completion of a
satisfactory three-dimensional structure of theta.
As part of a program aimed at understanding the molecular mechanism
of the core, we have set out to investigate the association of the theta
and e subunits. Preliminary experiments,
in which 15N, 13C-labelled theta
is bound to the unlabelled N-terminal domain of the e
subunit, have demonstrated that more 1H-15N
crosspeaks with a greater dispersion are observed in the 15N
HSQC spectrum of the complex. A partially refined structure of the theta
subunit bound to e has been determined.
The basic structure of theta has not changed
but two of the helices that were poorly defined in the uncomplexed theta
subunit are properly formed in the complex. We have recently mapped
the binding surface of e on theta
using advanced NMR techniques. Not surprisingly, the surface
corresponds to a hydrophobic patch on theta
formed on one of the previously ill-formed a-helices.
Work is now in progress to align the refined NMR structure of theta
with the X-ray structure of the N-terminal half of e.
This will constitute a rare combination of these two powerful
structural technologies (with E.M. Bulloch, N.E. Dixon,
S. Hamdan, T.K. Ronson, S.E. Brown
[Entomology CSIRO])
Structures of Small Chaperone Proteins
Small
heat shock proteins stabilise other proteins under conditions of
stress, for example, heat, oxidation, or the presence of heavy
metals. The nature of the chaperone action of these proteins is
poorly understood. Although these proteins are small in mass, they
exist as large oligomeric species and, hence, present a challenge to
NMR techniques. Two small heat shock proteins, hsp18.1 and hsp16.9,
have been overexpressed and uniformly labelled with 15N in
the laboratories of Associate Professor John Carver and Professor
Elizabeth Zierling. The spectra of hsp18.1 were disappointing, but
the results with hsp16.9 are very encouraging and analyses of the
spectra are now in progress. (with J.A. Carver, R.A. Lindner
[U. Wollongong], E. Zierling [U. Arizona, Tucson])
NMR Studies of the Interaction of Spermine with Oligonucleotides
Spermine,
an aliphatic polycationic molecule found in all cells, has an
essential role in cell growth and differentiation. At present, there
is no thorough understanding of how polyamines exert their
physiological effects. Spermine is known to interact with both DNA
and with proteins, yet the details of these interactions and the
molecular basis of the biological function of spermine are poorly
understood. There is evidence in the literature that spermine
interacts with different forms of DNA in distinct and divergent
modes. We have confirmed this and have characterised the complexes
of spermine with duplex B-DNA and G-DNA using a specifically
13C-labelled spermine and advanced NMR techniques to take
advantage of the specific isotope label on spermine. 13C
T1 and T2 relaxation times and changes in the
1H-13C dipolar coupling of 13C-labelled
spermine are used to characterise the dynamics of spermine in the
presence of different forms of DNA. Spermine is bound more tightly
to G-DNA than B-DNA. We have recently located this unique
interaction of spermine with G-DNA to the loop regions of folded DNA
quadruplexes. (with J. Coughlan)
ESX, a Protein Overexpressed in the Early Stages of Epithelial Breast
Cancer
ESX
is a protein that belongs to the Ets family of transcription
factors. Ets proteins exhibit diverse roles in development,
cell differentiation and tissue-specific gene expression and are
implicated in cancers such as acute myeloid leukemia and Ewings
sarcoma. The ESX transcription factor may have a role in the
activation of the HER2/neu oncogene, which is overexpressed in over
40% of breast tumours. We are interested in determining the
structure of ESX using X-ray crystallography and NMR. To this end we
have overexpressed the C-terminal end of ESX containing the two
DNA-binding domains. Attempts will be made to crystallize this
fragment. A 15N-labelled form of the C-terminal
fragment of ESX is also available and NMR studies will commence
shortly. The long-term goal is to determine the complete structure
of ESX and studies to optimize expression and folding of the
N-terminal fragment and the complete protein are envisaged.
This project is supported in part by a Yamagiwa-Yoshida travel grant
from the International Union against Cancer. (with C.C. Benz,
G. Scott [Buck Institute for Age Research, USA])
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