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Research
The
central theme of our research is the application of nuclear
magnetic resonance (NMR) spectroscopy to the solution of
biochemical problems. The unique power of NMR lies in its ability to
provide detailed chemical and structural information at an atomic level
about molecules in solution -- even when they are present in living cells
or organisms.
The general strategy is to use multidimensional (2D, 3D, and
4D) multinuclear magnetic resonance techniques to detect and assign
resonances from atoms of biological interest (e.g. 1H, 13C, 15N, and 31P).
With these assignments in hand, we can then interpret the wealth of
spectral information present in coupling constants, relaxation rates,
cross-relaxation rates, and chemical shifts. Proton-proton
cross-relaxation rates and a variety of measured coupling constants are
used to derive three-dimensional structures of these macromolecules.
Relaxation rates, line-shapes, and Nuclear Overhauser Effect measurements
provide information about molecular motions and conformational changes.
The kinds of information gained from such investigations can be critical
for learning how these molecules work and how they can be redesigned to
have desired properties.
We exploit recombinant DNA technology as a means
for producing the large amounts of protein needed for NMR investigations
and for introducing stable isotopes of interest (most commonly 2H, 13C,
and 15N). Mutagenesis studies allow us to test hypotheses about the roles
of individual amino acid residues in determining properties such as local
structure, conformations and mobilities of side chains, hydrogen exchange
kinetics, rates of protein folding or unfolding, pKa values,
oxidation-reduction potentials, and ligand binding.
In particular, our work focuses on the following topics:
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(i)
Modulation of iron-sulfur cluster
properties
Iron-sulfur proteins participate in many
biochemical processes including electron transfer, substrate binding and
catalysis, and regulation and sensing. As their name implies, these
metalloproteins contain one or more iron ions ligated mostly, though not
exclusively, by sulfur atoms. The immediate environment of the metal
ion(s) has a significant impact on its properties but the details of this
phenomenon are poorly understood. Through studies on Clostridium pasteurianum rubredoxin and selected [2Fe2S]
ferredoxins we hope to advance our understanding of how sequence and
structure determine the properties of iron ions in iron-sulfur
proteins. |
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(ii)
Brazzein - a sweet-tasting protein
Found in a variety of African and South
Asian fruits, sweet-tasting proteins are perceived sweet by humans and
old-world monkeys. In the last 30 years six such proteins have been
discovered -- they all have different molecular lengths, as well as very
little sequence and structural homology. Despite significant research
efforts, the features responsible for the sweet taste of these proteins
are still very poorly understood. Our work focuses on brazzein, a sweet
protein of 54 amino acid residues, and its interaction with sweet taste
receptors. |
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(iii)
Acyl carrier protein
Acyl carrier protein is a small protein
involved in the movement of various substrates between enzymes in
biosynthetic pathways. One of its functions involves carrying unsaturated
long chain fatty acids to the enzyme delta-9-desaturase which introduces a
double bond between C9 and C10 of the fatty acid chain. Work in the lab
concentrates on the structural characterization of acyl carrier protein.
Significant effort is also devoted to advance our understanding on how
acyl carrier protein binds to delta-9-desaturase. |
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(iv) Biogenesis of iron-sulfur clusters
Iron-sulfur clusters are ubiquitous in
nature and participate in many biochemical processes. As incorrect
cluster assembly can result in disease, it is imperative portant to
understand the mechanism behind steps leading to such problems. We
study three proteins involved in the biogenesis of iron-sulfur clusters:
IscU, HscA and HscB. Available experimental evidence strongly
suggests that IscU acts as a scaffold for the assembly and subsequent
transfer of Fe/S clusters to target apo-proteins. The details of
these steps, however as well as the roles of HscA and HscB in the cluster
transfer process are not well understood. We are using NMR
spectroscopy to gain novel insight into these unexplored areas of Fe/S
biochemistry. |
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(v) Automation in NMR investigation of protein structure
Alternately,
graduate students and postdoctoral fellows in the laboratory may focus on
developing instrumentation or novel ways of collecting or analyzing NMR data.
Current
developed software and techniques in our laboratory include:
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PISTACHIO: A probabilistic
approach to chemical shift assignment
- HIFI-NMR:
Fast data collection and processing
of multidimensional NMR
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PECAN: Secondary structure
prediction of proteins
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LACS: Detection of outlier
chemical shifts
- ALMONDS:
Chemical shift prediction based on sequence
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