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Principal Investigator: Ian Bakanas
Organization: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Fiscal Year: 2024
Award: $73,828
Funding agency: National Institute of General Medical Sciences
Project Summary/Abstract
The goal of this proposal is to elucidate biological mechanisms for proton transfer by designing function from
scratch. The coupled movement of protons and electrons is crucial to biological energy transduction and central
to life. While electron transfer (ET) has been extensively studied, less is known about the corresponding proton
transfer (PT) due to lack of easily observable experimental readouts. Computational protein design enables us
to study these phenomena in a ground-up manner where a protein scaffold can be designed from first principles
to mimic biological function in isolated and experimentally tractable ways. This proposal centers on the binding
of abiological photoacid cofactors that would give distinct spectroscopic readouts for PT as a function of distance.
The electron-deficient metal porphyrin photoacid cofactors used in this proposal are characterized by dramatic
acidification upon photoexcitation and distinct spectroscopic changes upon deprotonation. These cofactor
properties combined with our lab’s history of success in the design of porphyrin-binding proteins make them ideal
for use in this proposal. Using computational tools recently developed in the DeGrado lab (vdMs and COMBS),
the cofactor will be positioned within a designer protein scaffold H-bonded to a proton-accepting residue. This
will enable the spectroscopic study of proton on-off rates upon irradiation and subsequent deprotonation of the
cofactor. These ligand-binding proteins will be experimentally characterized through X-ray crystallography and
NMR experiments to validate the proposed structure and binding mode. Ultrafast absorbance spectroscopy
experiments will be carried out by our long-term collaborators in the Therien lab at Duke University. Following
characterization, the proton-accepting residue will be iteratively moved down the protein scaffold with a designed
“water-wire” in its wake to allow spectroscopic observation of the proton movement over varying distances.
Further, a second cofactor binding site will be built to bind a pH-responsive dye. This will allow for end-to-end
monitoring of PT with measurable readouts in a protein system for the first time. This research will significantly
advance our understanding of biological proton dynamics, critically test our ability to design ligand-binding
proteins, push toward the intentional design of water wires, and innovate a new strategy for the design of proteins
that bind multiple interacting cofactors. The use of computational design tools (Rosetta, COMBS, RFdiffusion,
ProteinMPNN, Alphafold), as well as routine protein expression, purification, and characterization will fulfill the
training goals of my postdoctoral tenure, combining my skills in organic synthesis with protein design. Together
these skills will prepare me for an independent research career focused on the design of functional proteins and
enzymes to catalyze new-to-nature reactions.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Acidity><Affinity><Amino Acid Sequence><Area><Back><Bacteria><Binding><Binding Proteins><Binding Sites><Biological><Biological Function><Biological Process><COVID-19 virus><COVID19 virus><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Charge><Chemicals><CoV-2><CoV2><Collaborations><Coloring Agents><Combining Site><Complication><Computational toolkit><Coupled><Coupling><Device Designs><Dorsum><Drugs><Dyes><Electron Transport><Electrons><Engineering><Enzyme Gene><Enzymes><Generations><Goals><H+ element><H-bond><Harvest><High Prevalence><History><Hydrogen Bonding><Hydrogen Ions><Hydrogen Oxide><Influenza Virus><Intracellular Communication and Signaling><Kinetics><Life><Ligand Binding Protein><Ligand Binding Protein Gene><Light><Measurable><Medication><Membrane><Metals><Methodology><Methods><Molecular Interaction><Monitor><Movement><NMR Spectrometer><NMR Spectroscopy><Nature><Negative Beta Particle><Negatrons><Network-based><Organelles><Organic Synthesis><Oxidation-Reduction><Pathway interactions><Pharmaceutical Preparations><Photoradiation><Photosynthesis><Porphyrins><Position><Positioning Attribute><Postdoc><Postdoctoral Fellow><Primary Protein Structure><Process><Property><Protein Binding><Protein Engineering><Proteins><Protons><Pump><Reaction><Reactive Site><Recording of previous events><Redox><Reproduction><Research><Research Associate><Rhodopsin><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Sampling><Scaffolding Protein><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Signal Transduction><Signal Transduction Systems><Signaling><Single Crystal Diffraction><Solvents><Spectroscopy><Spectrum Analyses><Spectrum Analysis><Structure><System><Testing><Therapeutic><Time><Training><Universities><Viral><Visual Purple><Water><Wuhan coronavirus><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><absorption><biologic><biological signal transduction><biological systems><body movement><bound protein><career><cofactor><computational toolbox><computational tools><computational toolset><computerized tools><coronavirus disease 2019 virus><coronavirus disease-19 virus><deprotonation><design><designing><drug discovery><drug/agent><electron transfer><experiment><experimental research><experimental study><experiments><generative models><genetic protein engineering><hCoV19><histories><influenzavirus><innovate><innovation><innovative><insight><irradiation><membrane structure><migration><nCoV2><neural network><new approaches><novel approaches><novel strategies><novel strategy><nuclear magnetic resonance spectroscopy><oxidation reduction reaction><pathway><post-doc><post-doctoral><post-doctoral trainee><protein complex><protein design><protein expression><protein folding><protein sequence><protonation><rational design><research associates><skills><small molecule><spectroscopic study><spectroscopic survey><success><tool>