Sensitive detection of protein binding to the plasma membrane with dual-color Z-scan fluorescence

I. Angert, S. R. Karuka, J. Hennen, Y. Chen, J. P. Albanesi, L. M. Mansky, J. D. Mueller

Research output: Contribution to journalArticlepeer-review

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 co-expression of a second distinctly colored fluorescent protein provides a soluble reference species, which 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. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2α but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of Kd ~16 µM and Hill coefficient of n ~2.

Original languageEnglish (US)
JournalUnknown Journal
DOIs
StatePublished - Sep 13 2019

Keywords

  • Brightness
  • Cooperativity
  • Fluorescence
  • Langmuir-Isotherm
  • Peripheral membrane protein
  • Protein-lipid interactions

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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