Synthetic Biology-Based Solution NMR Studies on Membrane Proteins in Lipid Environments

Methods Enzymol. 2019;614:143-185. doi: 10.1016/bs.mie.2018.08.019. Epub 2018 Dec 21.

Abstract

Although membrane proteins are in the focus of biochemical research for many decades the general knowledge of this important class is far behind soluble proteins. Despite several recent technical developments, the most challenging feature still is the generation of high-quality samples in environments suitable for the selected application. Reconstitution of membrane proteins into lipid bilayers will generate the most native-like environment and is therefore commonly desired. However, it poses tremendous problems to solution-state NMR analysis due to the dramatic increase in particle size resulting in high rotational correlation times. Nevertheless, a few promising strategies for the solution NMR analysis of membrane inserted proteins are emerging and will be discussed in this chapter. We focus on the generation of membrane protein samples in nanodisc membranes by cell-free systems and will describe the characteristic advantages of that platform in providing tailored protein expression and folding environments. We indicate frequent problems that have to be overcome in cell-free synthesis, nanodisc preparation, and customization for samples dedicated for solution-state NMR. Detailed instructions for sample preparation are given, and solution NMR approaches suitable for membrane proteins in bilayers are compiled. We further discuss the current strategies applied for signal detection from such difficult samples and describe the type of information that can be extracted from the various experiments. In summary, a comprehensive guideline for the analysis of membrane proteins in native-like membrane environments by solution-state NMR techniques will be provided.

Keywords: Backbone assignment; Bicelles; Cell-free protein expression; Isotopic labeling; Lipid bilayer; Membrane proteins; Nanodiscs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Directed RNA Polymerases / genetics
  • DNA-Directed RNA Polymerases / metabolism
  • Escherichia coli / chemistry*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Lipid Bilayers / chemistry*
  • Lipid Bilayers / metabolism
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism
  • Nanostructures / chemistry
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Phosphorylcholine / analogs & derivatives
  • Phosphorylcholine / chemistry
  • Phosphorylcholine / metabolism
  • Protein Folding
  • Sodium Cholate / chemistry
  • Sodium Cholate / metabolism
  • Subcellular Fractions / chemistry
  • Subcellular Fractions / metabolism
  • Synthetic Biology / methods*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism

Substances

  • Lipid Bilayers
  • Membrane Proteins
  • Viral Proteins
  • Phosphorylcholine
  • dodecylphosphocholine
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • Sodium Cholate