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Department of Plant and Microbial Biology 
Microbiology Graduate Programs

Paul Hamilton

PH
Paul Hamilton - Headshot for Directory

Emeritus Associate Professor

Emeriti

Department of Plant and Microbial Biology

Bio

As we enter a post-genomic era, it has become apparent that a significant number of the genes identified in bacterial genomes encode proteins of unknown function.  The development and refinement of tools and techniques to elucidate the function of these poorly characterized proteins will be required.  My research interests are in developing peptide-based tools to determine the role of various proteins of unknown function in bacterial metabolism and virulence.  In addition, many of the genes encoding proteins of unknown function are required for cell viability.  These essential genes represent valid targets for antibacterial drug discovery but are typically not accessible using standard biochemical assays.  The peptide-based tools developed to ascribe function to these essential gene products can be readily converted into high throughput screening (HTS) assays for drug discovery.

Determining the Activity for Bacterial Proteins of Unknown Function

Over 700 bacterial genomes have been completely sequenced and at least an equal number are in progress.  This work provides a large amount of genetic and genomic information; however, to extend the value of these data will require functional characterization of the gene products.  Mycoplasma genitalium has one of the smallest genomes of any free-living organism.  Characterization of the genes essential for M. genitalium viability has shown that 382 of the 482 M. genitalium protein-coding genes are essential. Of those 382 essential genes, 28% encode proteins of unknown function.  To take full advantage of the bacterial genomic information available and to have a more complete understanding of the role these proteins of unknown function in bacterial physiology will require additional tools.  High affinity binding reagents and inhibitors have traditionally been used as tools to characterize protein function.  Peptide-based phage display technology offers a way to rapidly isolate such reagents.

Phage display is a powerful technology that my lab has used to identify peptides that bind to a wide range of targets from proteins to biomaterials.  Combinatorial peptide libraries are constructed by inserting peptide-encoding oligonucleotides into gene III of bacteriophage M13.  The resulting peptide fusions are displayed on the surface of the bacteriophage and can be screened for binding to a target of interest.  When applied to protein targets, we have observed that the peptides are directed to functional sites on the protein and don’t bind randomly to the protein.  For an enzyme, this includes targeting the active site and the peptide will function as an inhibitor of the enzymatic activity, even if the enzymatic substrates are none peptidic in nature.  For example, we have isolated peptides that bind to alcohol dehydrogenase and inhibit the conversion of ethanol to acetaldehyde with a Ki of 80 nM.

In addition to their use as inhibitors in biochemical reactions, peptides can be expressed inside cells to bind the target protein and disrupt its function.  When applied to proteins that are essential for cell viability, this “protein knockout” will result in inhibition of cell growth.  We have validated this protein knockout approach with several essential bacterial proteins and want to apply it to proteins of unknown function that are indentified by genomics.

Antibacterial Drug Discovery

Infectious diseases are still a leading cause of death worldwide.  With increasing antibiotic resistance among bacterial pathogens and the emergence of hard-to-treat opportunistic infections, the need for new antibiotics continues.  Among pharmaceutical companies, however, antibacterial research and development has been downsized or eliminated.  Bacterial genomics has provided a large number of new targets for antibacterial drug discovery.  As described above, a significant number of the essential genes identified by genomics encode proteins of unknown function.  In previous work, we have shown that peptides isolated by phage display can be used as surrogate ligands to develop HTS-compatible assays to screen chemical compound collections for the discovery of new antibacterial agents.

My longer term goals include elucidating the role of proteins of unknown function in bacterial metabolism and virulence and developing small chemical molecules and peptides as research tools for characterization of bacterial proteins and enzymes.

Area(s) of Expertise

Microbial biotechnology, bacteriophage biology, biotechnology applications of thermophiles, phage display technology

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