A Strategic Approach for Fluorescence Imaging of Membrane Proteins in a Native-like Environment

Cell Chem Biol. 2020 Feb 20;27(2):245-251.e3. doi: 10.1016/j.chembiol.2019.11.008. Epub 2019 Dec 9.

Abstract

Biological membranes are complex barriers in which membrane proteins and thousands of lipidic species participate in structural and functional interactions. Developing a strategic approach that allows uniform labeling of membrane proteins while maintaining a lipidic environment that retains functional interactions is highly desirable for in vitro fluorescence studies. Herein, we focus on complementing current methods by integrating the powerful processes of unnatural amino acid mutagenesis, bioorthogonal labeling, and the detergent-free membrane protein solubilization based on the amphiphilic styrene-maleic acid (SMA) polymer. Importantly, the SMA polymer preserves a thermodynamically stable shell of phospholipids. The approach that we present is both rapid and generalizable providing a population of uniquely labeled membrane proteins in lipid nanoparticles for quantitative fluorescence-based studies.

Keywords: SMALP; bioorthogonal labeling; detergent-free solubilization; membrane protein; non-canonical amino acid mutagenesis; single-molecule fluorescence imaging; styrene-maleic acid copolymer.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Amino Acids / metabolism
  • Amino Acyl-tRNA Synthetases / chemistry
  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Escherichia coli / metabolism
  • Fluorescent Dyes / chemistry
  • Maleates / chemistry
  • Maleates / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microscopy, Fluorescence
  • Mutagenesis, Site-Directed
  • Nanoparticles / chemistry
  • Phospholipids / chemistry
  • Polystyrenes / chemistry
  • Polystyrenes / metabolism
  • Single Molecule Imaging / methods*
  • Solubility

Substances

  • Amino Acids
  • Bacterial Proteins
  • Fluorescent Dyes
  • Maleates
  • Membrane Proteins
  • Phospholipids
  • Polystyrenes
  • styrene-maleic acid polymer
  • Amino Acyl-tRNA Synthetases