title: Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings.
abstract: The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid-protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (K D ) and kinetics (kon and koff ). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached.
authors.affiliation: Department of Physics, Florida State University, Tallahassee, FL, 32306-4350, USA.
authors.forename: Troy W
authors.initials: TW
authors.lastname: Lowry
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306-4380, USA.
authors.forename: Hanaa
authors.initials: H
authors.lastname: Hariri
authors.affiliation: Department of Physics, Florida State University, Tallahassee, FL, 32306-4350, USA.
authors.forename: Plengchart
authors.initials: P
authors.lastname: Prommapan
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL, 32306-4370, USA.
authors.forename: Aubrey
authors.initials: A
authors.lastname: Kusi-Appiah
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL, 32306-4370, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Vafai
authors.affiliation: Department of Biomedical Science, College of Medicine, Florida State University, Tallahassee, FL, 32306-4300, USA.
authors.forename: Ewa A
authors.initials: EA
authors.lastname: Bienkiewicz
authors.affiliation: Department of Physics, Florida State University, Tallahassee, FL, 32306-4350, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Van Winkle
authors.affiliation: Institute of Molecular Biophysics and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32306-4380, USA.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Stagg
authors.affiliation: Department of Biological Science and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306-4370, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Lenhert
mesh_terms: D007700:Kinetics; D008027:Light; D008051:Lipid Bilayers / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D055620:Optical Phenomena
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins
keywords: biosensors; kinetics; lipid multilayer gratings; lipids; membranes; microarrays