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Structural
Biology & Proteomics Research
Proteomics
The completion of the Human Genome Project has heralded
the beginning of a much more formidable task, that of defining the
structures and functions of about 100,000 human proteins, the gene
products. Protein Science will become the most important discipline
as protein structurefunction relationships, folding and stability,
regulation through post-translational modifications, and their interaction
with other molecules including DNA, RNA, proteins and small ligands
are important for biological functions. Thus, attention is now increasingly
shifting towards the identification and characterization of PROTEin
products expressed by the genOME of an organism. The term 'PROTEOME'
is used to describe this approach of studying the gene products
and their functions. Proteomics (proteome analysis) is the study
of protein properties (expression level, post-translational modifications,
interactions etc) on a large-scale to obtain a global, integrated
view of disease processes, cellular processes and networks at the
protein level. It is now feasible to analyze the presence and level
of proteins expressed in cells, tissues or whole organisms. Proteins
are resolved as spots using 2Dgel electrophoresis and these spots
are characterized using mass spectrometric analyses of their tryptic
digests in MALDITOF and ESI tandem mass spectrometers. Based on
the 'mass-map' (or 'mass-fingerprint') and 'sequence-tag' searches
in protein sequence databases, these spots are identified. The alteration
of the protein profiles upon perturbation by different signals,
viral infections, and changes of metabolic, environmental or hormonal
influences can be monitored and quantitated. Thus, proteomics is
providing a powerful tool to identify proteins that are potential
targets for therapeutics/interventions. The Lee Hiok Kwee Functional
Genomics Laboratories (FGL, an open laboratory at Blk. S3 level
2) and the Protein and Proteomics Center (PPC, a core facility at
Blk. S3 level 3) in the Department of Biological Sciences are major
initiatives in the area of proteomics. The PPC plays a vital role
by providing a wide variety of advanced equipment, e.g. the ABI
Proteomics Analyzer MALDI-Tof-Tof mass spectrometer, expertise and
training to an array of research projects in structural biology
and proteomics currently underway at DBS and other NUS life sciences
departments and research institutes. In addition, the Department
will have its own inhouse bioinformatics facilities at the Computational
Biology Laboratories (CBL at Blk. S3 level 1) in the near future,
to provide bioinformatics training and resource support to DBS researchers
and students.
Several researchers at DBS are
actively pursuing studies in various areas of proteomics and functional
genomics. These include bacterial pathogenesis, allergenic components
and allergen-specific immune responses, proteomics and structural
biology of marine virus, transgenic fish, proteomics in developmental
biology of zebrafish, molecular endocrinology of vertebrates, and
liver and colorectal cancer proteomics. The next generation of protein
microarray technologies are also being developed, promising the
ability to conduct analyses of tens of thousands of proteins simultaneously.
Structural Biology
Proteins that are identified as potential targets for protein engineering
and intervention through proteomic approaches are being expressed
in larger quantities for structural and activity/interaction studies.
Understanding the role played by a protein molecule requires a detailed
picture of its three-dimensional (3-D) structure and identification
of crucial amino acid residues involved in its interaction with
targets such as other protein molecule, peptide, DNA, RNA and small
molecule ligands. Nuclear Magnetic Resonance (NMR) spectroscopy,
X-ray crystallography and cryo-electron microscopy are three techniques
that can provide protein structures. Over the past several years
multi-dimensional (3-D or 4-D), multinuclear (1H, 15N and 13C) solution
NMR spectroscopy combined with protein labeling (2H) strategy has
become a powerful technology for obtaining both structural and dynamic
information on protein and protein-ligand systems with molecular
weight up to 30-40 kDa. NMR spectroscopy can solve protein and peptide
structures in solution, thus protein structure and dynamics under
physiological conditions can be investigated. NMR spectroscopy can
also be used for drug screening and discovery; diagnosisof human
diseases through analysis of biological fluids; and identification
of active components in complex natural products. The use of NMR
spectroscopy, however, is limited by the molecular weight of the
proteins that can be studied. Large protein molecules usually face
the problem of serious resonances overlap and loss of signal intensity
due to fast relaxation. The problem can be partly alleviated by
optimizing NMR pulse-sequence and the use of a stronger magnet.
The existing 500MHz NMR spectrometer at the Chemical and Molecular
Analysis Center was upgraded with a 4-channel cryoprobe for high-resolution
protein structure determination. Very recently, the 800 MHz ultra-shielded,
ultra-stabilized NMR spectrometer was installed as a core facility
for the Life Sciences Initiative. The 800 MHz spectrometer offers
significantly improved sensitivity and unprecedented resolution,
thus speeding up the structure determination process by reducing
resonances overlap and data acquisition time. This is essential
for proteomics study because it greatly extends the size range of
the numerous proteins that can be studied by NMR.
X-ray crystallography is another
method that pictures the macromolecular structures at atomic resolution.
The protein is over-expressed in a suitable system and purified
to homogeneity. It is crystallized and diffraction data are collected
using a detector mounted on rotating anode X-ray generator or synchrotron.
The structure is solved by established crystallographic methods
such as Multiple Isomorphous Replacement (MIR) method and Multiwavelength
Anomalous Dispersion (MAD) method and the electron density map is
drawn. The protein model is built based on the electron density
map and then refined. Structural information obtained through either
NMR or X-ray crystallography enables a lot of novel applications.
The high-resolution structures contribute significantly to our understanding
of genetic diseases. With the help of such precise structures, a
suitable ligand/ inhibitor can be designed through combinatorial
chemistry, computer modeling and docking techniques. The 'structure
based drug design' promises effective drugs for diseases in the
near future. The Lee Wee Kheng Structural Biology Laboratories (SBL,
an open laboratory at Blk. S3 level 4) house a total of five research
groups (three NMR and two X-ray crystallography) actively pursuing
structural/functional relationships of various bio-medically important
proteins.
Another increasingly used technique
in structural biology is cryo-electron microscopy (cryo-EM). Cryo-EM,
while not yet yielding individual structures at resolutions as high
as those obtained with NMR or X-ray crystallography, is readily
applicable to macromolecular complexes or less soluble membrane
proteins that are difficult to crystallize. If the structures of
the individual components are known to a high resolution, a cryo-EM
density map of the entire macromolecular complex in the intermediate
resolution range of 15-30 Å, will usually allow their relative positions
and orientations to be specified to within a few angstroms, thus
identifying the interaction surfaces. Such interaction information
cannot, in general, be inferred from the structures of the individual
components even at very high resolution. This information, however,
is essential for a mechanistic understanding of biological functions,
as most cellular functions are carried out through formation of
large complexes.
NMR and X-ray crystallography
along with cryo-EM will help solve three-dimensional structures
of proteins and protein complexes of all sizes. When needed, pure
proteins are obtained using various expression systems, such as
microorganisms, viruses, as well as plant, mammalian and insect
cells. We also have the capability to synthesize large proteins
through peptide chemistry with unnatural amino acid functionalities.
Alternatively, rather than generating the whole protein, peptides
corresponding to different regions of a protein target can be generated
for protein interaction studies. The department is fully equipped
with facilities to study protein-protein or protein-peptide interactions
using biophysical methods, NMR titration methods, protein based
biosensor and isothermal titration calorimetry. Experimental binding
data together with macromolecular modeling will shed light on the
detailed mechanisms of binding between a protein molecule and its
target ligand.
We are currently determining the
structures of several proteins including toxins from snake venoms,
bacterial type III secretion system proteins, SARS viral proteins
and dust mite allergens. We are also examining the dynamic folding
and interaction of fatty acid binding protein, phospholipase A2
and its inhibitor, endotoxin-binding peptides, and amyloid peptides.
Our research interests include also structure-function relationships
and protein design and engineering.
The department is also building
a strong program in Combinatorial Biology and Combinatorial Chemistry,
which will be the basis of setting up high-throughput platforms
for drug discovery and design. The department is equipped with combinatorial
organic synthesizers capable of running 20 to 400 parallel syntheses
of biomolecules such as peptides, oligonucleotides and
drug-like small molecules. Currently, peptide and nonpeptide libraries
are being designed, synthesized and screened for activities against
a whole range of enzymes such as proteases, kinases and so on. New
biochemical and biological assays, most of which are fluorescencebased,
are being developed to support the combinatorial capacity. Using
techniques in Combinatorial Biology, novel approaches are also being
developed, within the department, that are capable of simultaneous
expression and purification of tens of thousands of proteins, in
vitro. In addition, advanced robotic systems are being set up that
are capable of spotting up to 10,000 different biomolecules, each
in nL-size droplets, onto standard microscope glass slides (2"
x 1"). These 'miniaturalized' biochips will be used as next-generation
tools for proteomics, drug discovery and diagnostics.
For more details, see the personal
research profiles of F.T. Chew, J.L. Ding, C.L. Hew, R.M. Kini,
K.Y. Leung, H.Y.K. Mok, D.W. Yang, J.X. Song, K. Swaminathan, J.
Sivaraman and S.Q. Yao at
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