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Principal Investigator: MICHAEL L GROSS
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2023
Award: $465,693
Funding agency: National Institute of General Medical Sciences
Project Summary
Mass spectrometry (MS) based footprinting is emerging as a powerful means to answer biological questions
about membrane proteins (MPs), which participate in almost all physiological processes and represent more
than 60% of drug targets. This approach affords sufficient structural information for the dynamic, native
conformations and interactions of MPs in cells, which are beyond the reach of traditional structural methods (e.g.,
cryo-EM and crystallography). This bottom-up MS footprinting is complementary to but potentially more
informative than top-down native MS, which does not provide spatial resolution for MPs and is conducted in the
nonnative gas phase. Here we propose to continue development of novel MS footprinting methods in live cells
and native membranes. Our objective is to design, prepare, test, and improve chemical probes that provide high
footprinting coverage. We will then apply them to reveal drug interactions and cellular trafficking regulation of a
glucose transporter, GLUT1, a prominent anticancer drug target and a model MP representing ~ 25% of known
transport proteins. MS footprinting of MPs, however, poses three major challenges. 1) MPs, which are
hydrophobic and buried in lipid bilayers, are resistant to traditional probes (e.g., HDX, •OH radicals) that
penetrate poorly and give insufficient labeling. 2) Aliphatic side chains of transmembrane regions contain C–H
and C-C bonds that are unreactive with most chemical probes. 3) The footprinting needs to be conducted in cells
or membranes to maintain native conformation and interaction of MPs. Our hypotheses are: (1) Complementary
modifications of C-H and X–H bonds by free radicals produced photochemically and by nucleophilic reagents
maximize footprinting coverage. (2) Tuning the hydrophobicity of the reagents or their precursors allows access
to membrane-embedded MPs. (3) Novel membrane fusion techniques introduce inert footprinters into live cells
and native membranes for subsequent photoactivated footprinting. Our hypotheses are built on extensive
preliminary data. Three years of funding supported publication of 18 papers in high-profile journals. A significant
example describes laser activation of TiO2 nanoparticles attached to liposomes to generate high local
concentrations of radicals. Simultaneous membrane poration permits radical entry to footprint with sufficient
structural resolution that reports the ligand-binding sites and rocker-switch motions of GLUT1. Building on these
successes, we will pursue two specific aims: (1) develop new chemical probes for MS footprinting of MPs; and
(2) conduct comprehensive footprinting in native membranes and live cells to reveal anticancer drug interactions
and trafficking regulations of GLUT1. Our innovative footprinting coupled with bottom-up MS proteomics analysis
will establish bio-orthogonal footprinters that afford comprehensive coverage of both hydrophobic and hydrophilic
regions of MPs and reveal drug interactions and structural regulation of human MPs under inarguable native
settings. The impact of the proposed approach should readily expand because MS-based footprinting can be
broadly applied in structural proteomics to expedite drug discovery and structural studies of cellular processes.
Terms: <Active Oxygen><Anti-Cancer Agents><Antineoplastic Agents><Antineoplastic Drugs><Antineoplastics><Azides><Benchmarking><Benzophenones><Best Practice Analysis><Binding Sites><Biological><Cancer Drug><Carrier Proteins><Cell Body><Cell Function><Cell Isolation><Cell Membrane Lipids><Cell Membrane Permeability><Cell Process><Cell Segregation><Cell Separation><Cell Separation Technology><Cell Survival><Cell Viability><Cell physiology><Cells><Cellular Function><Cellular Metabolic Process><Cellular Physiology><Cellular Process><Chemical Structure><Chemicals><Chemistry><Combining Site><Consumption><Coupled><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallographies><Crystallography><Data><Development><Drug Interactions><Drug Targeting><Electron Cryomicroscopy><Ensure><Erythrocyte/Hepatoma Glucose Transporter><FAST Kinase><FAST gene><FAST protein><FASTK><FASTK Gene><FLJ13079><Fas-activated serine-threonine kinase><Free Radicals><Funding><GLUT><GLUT1><Gases><Glucose Binding Protein><Glucose Transport Protein><Glucose Transporter><Glucose Transporter 1><Goals><Grant><H-bond><Human><Human Figure><Human body><Hydrogen Bonding><Hydrophobicity><Iodides><Iodine><Journals><Label><Laser Electromagnetic><Laser Radiation><Lasers><Ligand Binding><Lipid Bilayers><Lipids><Liposomal><Liposomes><Magazine><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Membrane><Membrane Fusion><Membrane Lipids><Membrane Protein Gene><Membrane Proteins><Membrane Transport Proteins><Membrane Transporters><Membrane-Associated Proteins><Methodology><Methods><Modern Man><Modification><Molecular Configuration><Molecular Conformation><Molecular Stereochemistry><Monitor><Motion><NIH><NSF attachment protein receptor><National Institutes of Health><Neoplastic Disease Chemotherapeutic Agents><Organism-Level Process><Organismal Process><Oxygen Radicals><Paper><Penetration><Permeability><Peroxides><Phase><Physiologic Processes><Physiological Processes><Pro-Oxidants><Process><Protein Dynamics><Protein Footprinting><Proteins><Proteomics><Public Health><Publications><R24><Reaction><Reactive Oxygen Species><Reactive Site><Reagent><Regulation><Reporting><Research><Research Resources><Resistance><Resolution><Resources><SLC2A1><SLC2A1 gene><SNAP receptor><SNARE><Scientific Publication><Series><Side><Solute Carrier Family 2, Facilitated Glucose Transporter, Member 1><Structure><Subcellular Process><Surface Proteins><TM Domain><Techniques><Technology><Testing><TiO2><Time><Transmembrane Domain><Transmembrane Region><Transport Protein Gene><Transport Proteins><Transporter Protein><Tumor-Specific Treatment Agents><United States National Institutes of Health><anti-cancer drug><anticancer agent><anticancer drug><benchmark><biologic><blood glucose regulation><carbene><cell metabolism><cell sorting><cellular metabaolism><conformation><conformational><conformational state><conformationally><conformations><cryo-EM><cryoEM><cryogenic electron microscopy><design><designing><developmental><drug candidate><drug discovery><glucose control><glucose homeostasis><glucose regulation><glucose-regulated proteins><hydrophilicity><improved><innovate><innovation><innovative><lipid bilayer membrane><lipid solubility><materials science><membrane model><membrane permeability><membrane structure><methylene><nano particle><nano-sized particle><nanodisk><nanoparticle><nanosized particle><non-Native><nonnative><novel><physical property><protein purification><protein structure><protein structures><proteins structure><reconstitute><reconstitution><resistant><resolutions><soluble N-ethylmaleimide-sensitive-factor attachment protein receptor><structural biology><success><titanium dioxide><titanium oxide><tool><trafficking>