Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence.

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence.
Biophys J. 2019 Dec 12;:
Authors: Angert I, Karuka SR, Hennen J, Chen Y, Albanesi JP, Mansky LM, Mueller JD
Abstract
Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2α...

Source: Biophysical Journal - December 11, 2019 Category: Physics Authors: Angert I, Karuka SR, Hennen J, Chen Y, Albanesi JP, Mansky LM, Mueller JD Tags: Biophys J Source Type: research