University of Minnesota
School of Physics & Astronomy

Physics and Astronomy Calendar

Thursday, November 21st 2019
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Isaac Angert, UMN (Mueller group)
Subject: Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence

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. In this talk I will give a brief overview of two-photon z-scan measurements of peripheral protein binding at the plasma membrane, which was previously developed by our group and successfully applied to quantify protein-PM interactions in living cells. I describe a dual color (DC) extension of the existing z-scan methods that utilizes co-expression of a second distinctly colored fluorescent protein. This second fluorescent species provides a soluble reference that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM binding measurements by an order of magnitude. Fit models for DC z-scan are developed and verified using simple model systems. We apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems, both relevant to human disease, for which existing z-scan techniques have proved inadequate. In particular, DC z-scan measurements of Human immunodeficiency virus type 1 (HIV-1) matrix protein indicate the presence of cooperative binding at the PM with a dissociation coefficient of K ~16 ┬ÁM and Hill coefficient of n ~2. I will discuss the relevance of these findings in the context of retroviral assembly as well as the future prospects for utilizing the new DC z-scan technique to quantify in-vivo protein-membrane engagement in wider-ranging systems.

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