- Research Overview
- Chemical Biology and Medicinal Chemistry
- Chemical Biology and Medicinal Chemistry Overview
- Discovering Enzyme Substrates and Functions
- Discovering Protein Ligands to Probe and Alter Function
- Discovering Enzyme Activators
- Analyzing Mechanisms of Drug Resistance via Chemical Biology
- Analyzing Enzyme Conformational Dynamics, Substrate Binding, and Catalysis
- Effective Drug Targeting of Pathogens via Medicinal Chemistry
- Computational Chemistry and Biology
- Computational Chemistry and Biology Overview
- Modeling protein regulation via allostery and post-translational modifications
- Visualizing and integrating bioinformatics and biomolecular data
- Modeling membrane permeation to optimize pharmacokinetics
- Determining enzyme function by predicting substrate specificity
- Physical Biology
- Protein and Cellular Engineering
- Protein and Cellular Engineering Overview
- Monitoring enzyme activity and disease biomarkers
- Generating human proteome antibodies via phage display and directed evolution
- Globally analyzing and dissecting apoptosis
- Proximity tagging of protein-protein interactions
- Investigating cellular interactions in tissues
- Creating fluorescent probes targeting the genome and key bio-pathways
- De novo design of catalytic and membrane proteins
- Probing and modulating membrane proteins
De novo design of catalytic and membrane proteins
Examples of our research and methods include
Designing metallo-proteins, membrane transporters
Having pioneered in de novo design of water-soluble alpha-helical bundles and synthetic membrane-spanning ion channels, department scientists have more recently computationally designed and genetically expressed self-assembling functional models of metalloproteins—both water soluble and transmembrane—that selectively bind specific hemes, unique non-natural porphyrins, and di-metal ion cofactors.
These designed proteins have included functional models of cytochromes and other proteins that transfer electrons across cellular membranes—a process that is, for example, key to the production of ATP, the molecule that transports energy to fuel the activities of life in our cells.
The design of model co-transporters from first principles is also ongoing. These transmembrane proteins use the gradient of one ion to drive the transport of another; for example, concentrating nutrients in a cell or removing toxins.
The design of model metalloproteins helps address key questions about how their abundant natural counterparts—up to a third of proteins employ metal in function— draw on a limited palette of amino acids and metal ions to produce a great diversity of catalytic functions. In particular, minimal scaffolds allow for focus on how changes to the active site yield that variety, imposing a specific geometry on bound cofactors and tuning their properties.
Likewise, the challenging engineering of transmembrane proteins, which play critical roles as channels, transporters, receptors, and thus pharmaceutical targets, tests emergent principles regarding the folding of proteins translocated to cell membranes. In the latter environment, hydrophobic forces in the cytosol give way to the assembly of linked subdomains via polar interactions and shape complementarity.
Such model proteins are also easier to systematically alter, incorporating non-natural cofactors, and to study, via spectroscopic probes, isotopic labels, and elements for surface immobilization.
Our applications of such protein engineering include
Reprogramming a protein via remodeling
Rationally reprogramming a de novo designed di-iron carboxylate protein by remodeling the substrate access cavity and adding a third histidine ligand to the metal binding cavity. The structural alterations changed the protein’s substrate (hydroquinones to arylamines), reaction type (two-electron oxidation to N-hydroxylation), and relative reaction rate (at least a million-fold increase).
These simple changes reprogram the reactivity of the resulting protein from hydroquinone oxidation to selective arylamine N-hydroxylation.
Synthesizing electron-transferring membrane proteins
Synthesizing a four-helix transmembrane metalloprotein complex that assembles without the hydrophobic driving force and binds two non-natural co-factors with high affinity and specificity. Dubbed PRIME (PoRphyrins In MEmbrane), the membrane protein positions two iron diphyenylporphyrins close enough to provide a pathway for electron transfer across the membrane bilayer.