Research Topics
Department of Chemistry
Ph.D. (1997) Cornell University
Research Topics
Research Interests
Microbial Proteomics. Bacterial genomes encode all possible virulence determinants, vaccine candidates, and potential drug targets. Further, a completed genomic sequence establishes a basis for high throughput analysis the proteins expressed (i.e., the proteome). Respiratory pathogens have been among the first to have their genomes entirely sequenced. Mycoplasma pneumoniae harbors the second smallest genome of any self-replicating life form and encodes 679 putative proteins. These genome-predicted proteins are being correlated with those actually present, detecting any biological event that generates a protein of different molecular composition than that predicted. These include sequence or reading frame errors, imprecise bioinformatics, co- or post-translational modifications, and mutational or proteolytic strategies for antigenic variation. Such an organism-wide protein analysis is also being performed on exotic bacteria like those found growing in near-boiling water. Instrumentation and BioInformatics. In addition to our commercial 4.7 Tesla instrument, we are currently building a 9.4 Tesla Fourier-Transform Mass Spectrometer (FTMS) at the National High Magnetic Field Laboratory in Tallahassee, FL; completion of this project in Fall of 2000 will outfit our lab with one of the highest performance mass spectrometers in the world. On the front end of these instruments, we are coupling capillary electrophoretic and chromatographic separations to enable submicrogram sampling and on-line analyses. Also, back end algorithms for computerized protein identification and correlation to raw genomic sequences will be required to achieve high protein analysis rates.
Natural Product Biosynthesis on MegaEnzymes. The common enzyme uses non-covalent interactions to bind and chemically alter the structure of its substrate. However, their are several families of enzymes that act upon their substrates via covalent catalysis, where building blocks of natural products are all bound covalently and then assembled on the enzyme. Such is the case with cyclosporin synthetase, the protein product of the largest gene known (48000 base pairs = 16000 amino acids). Due to the covalent attachment of all biosynthetic intermediates, we can measure the mass and relative abundances of them vs. time and "watch" the intermediates "crawl" along the enzyme as they grow. This will yield detailed insight into the mechanism(s) of non-ribosomal peptide- and polyketide-biogenesis. The high performance of FTMS is critical for extracting such kinetic data from complex proteolytic mixtures of large enzymes.
Summary. By searching for systems where protein sequence analysis can be translated into enzyme functional or mechanistic insight, our studies will result in understanding of basic microbiology, identification of new antimicrobial targets, and development of analytical instrumentation with unparalleled figures of merit. More generally, we are developing tools and generating data complementary to DNA sequencing and mRNA profiling in the new era of biology.
Key Words Electrospray Ionization, Fourier-Transform Mass Spectrometry,
capillary separations, Recombinant DNA, Posttranslational Modifications, Cotranslational,
infectious microbes, polyketides, non-ribosomal peptide synthesis, proteomics, protein
identification by database retrieval.
Current Research Funding
startup funds, Dreyfus, Searle, NIH pending (NIAID).