Research Overview
Our group's research focuses on structural characterization of membrane proteins and de novo protein design in order to understand biological processes relevant to human disease and develop novel therapeutics.
Protein Design
Proteins catalyze countless vital physicochemical reactions. Proteins do this by coordinating the substrates in specific, three-dimensional orientations, so understanding the structure is essential to understanding the function, disease etiology and drug design. Our lab uses a de novo design approach to explore the principals that govern folding, protein-cofactor, and protein-protein interaction, as well as subsequent functions, for both water- and lipid-soluble proteins. We then use various biophysical methods to define the success of our designs. Please, check out our de novo metalloprotein, DF2, featured as the Molecule of the Month on the PDB.
Antimicrobials
The rise of multidrug resistance is an alarming health concern in both nosocomial and community-acquired bacterial infections. We have designed small molecule mimics of antimicrobial peptides (evolutionarily conserved components of innate immunity in higher organisms), which display potent and selective activity against a broad spectrum of pathogens. The lead compound is currently in phase II clinical trials against multi-drug resistant Staphylococcal infections. We study the mechanism of action of these mimetics and the bacterial response to the presence of sub-inhibitory concentrations of these agents. We are also using these compounds as a tool for studying bacterial signaling systems involved in drug resistance and virulence.
Influenza
We are interested in the M2 protein of influenza because it is both a drug target and a model system for proton transport. M2 is a proton channel that is the target of a class of drugs called the adamantanes, though viral mutations have caused adamantane-resistant strains of influenza to become prevalent. M2 is also one of the smallest proton channels found in nature, since it is a homotetramer, with a minimally functional monomer length of only 25 amino acids. We perform structural studies on this system using NMR and crystallography, and we also create new M2 inhibitors targeting M2 then test their effectiveness.
HIV
A vaccine candidate that elicits broadly neutralizing monoclonal antibodies that confer immunity to HIV is yet to be discovered, but increasing data suggest that developing an effective vaccination is possible. In particular, the membrane proximal external region (MPER) of HIV-1's gp41 envelope protein is a promising target. The MPER is a highly conserved, dynamic region of gp41, exposed during the transient, pre-fusion state while the virus is binding to host T-cells and initiating fusion. A successful anti-MPER vaccine will elicit antibodies that recognize the MPER in this pre-fusion state, halt fusion, and render the virus incapable of reinitiating fusion. Using existing structural information on the HIV MPER and related viral proteins, we are designing vaccine constructs and working with collaborators to test their immunogenicity in rabbits. These design efforts would greatly benefit from characterization of the pre-fusion structure of the MPER, which is another goal of this project.
Alzheimer's Disease
Alzheimer's Disease is a devastating neurodegenerative disease that cruelly strips patients of memory, cognitive ability, and independence. Within the brains of patients, the A? peptide accumulates into fibrillar plaques that disrupt neuronal networks and soluble oligomeric complexes that are highly cytotoxic. However, relatively little is known about the protein conformations adopted by A? during disease pathogenesis. We are using biophysical techniques such as NMR to characterize protein conformation of oligomers and to identify structural polymorphisms in fibrils, as well as protein design to create novel disruption strategies that prevent these dangerous peptide accumulations.
Integrin Inhibitors
Integrins are heterodimeric transmembrane proteins that play a pivotal role in the signaling pathways that regulate processes as diverse as cell proliferation, differentiation, apoptosis, and cell migration. In collaboration with Joel Bennett (UPenn), we study the mechanism of signal transduction of integrins such as integrin ?IIb?3, with a particular focus on the role played by the membrane-spanning regions of this protein. We have also developed small molecule inhibitors of integrin ?2?1 in the platelet collagen receptor and now are expanding our small molecule inhibitor research to integrin ?v?1 in collaboration with Dean Sheppard (UCSF).
Bacterial Sensing
The two-component system is an essential stimulus-response mechanism in bacteria. It consists of a transmembrane histidine kinase that senses the environment and a response regulator that mediates the cellular response. We study the protein structures and conformational dynamics associated with signal transduction via two-component systems. Our methodology includes in-cell studies on bacteria, biochemical assays on purified proteins, structural and biophysical studies by x-ray crystallography, H/D exchange mass-spectrometry, NMR, and EPR. This project is synergistic with new antimicrobial development as many bacteria use two-component systems to develop antibiotic resistance.
Computational Tools
We use several computational approaches to assist us with designing proteins with desired properties. By efficiently searching the Protein Data Bank for backbone or amino acid motifs of interest, we can identify interactions frequently occurring in nature which can then be used as the foundation for our designs. Often we combine this nature-inspired approach with molecular modeling tools such as Rosetta and molecular dynamics simulations. Some of the applications we are pursuing with this approach is the design of peptides that can modulate protein-protein interactions, the design of transmembrane proteins with desired ion-transport properties, and the design of metal or small molecule binding sites.
For amino acid motifs, we use a tool recently developed in our lab, Suns, which has a built-in PyMol interface enabling the interactive building of motifs with the search results. For backbone motifs, we use MadCat, which efficiently compares the distance-map of a query motif to a database of pre-computed distance-maps for thousands of proteins from the PDB. Additionally, we have recently developed SuperCodons, a tool that allows the construction of a randomized DNA library that closely matches a chosen amino acid distribution. We have also developed the knowledge-based E(z)-3D Transmembrane Protein Orientation Potential, which predicts the most favorable orientation within a membrane for transmembrane proteins.
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