Quantification of Protein‐Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings |
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Authors: | Troy W. Lowry Plengchart Prommapan Aubrey Kusi‐Appiah Nicholas Vafai Ewa A. Bienkiewicz David H. Van Winkle Scott M. Stagg Steven Lenhert |
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Affiliation: | 1. Department of Physics, Florida State University, Tallahassee, FL, USA;2. Department of Biological Science, Florida State University, Tallahassee, FL, USA;3. Department of Biomedical Science, College of Medicine, Florida State University, Tallahassee, FL, USA;4. Institute of Molecular Biophysics and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA;5. Department of Biological Science and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA |
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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. |
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Keywords: | biosensors kinetics lipids membranes microarrays lipid multilayer gratings |
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