Ligand Binding in Allosteric Flavoproteins: Part 2. Quantitative Analysis of the Redox-Dependent Interaction of the Apoptosis-Inducing Factor (AIF) with Its Protein Partner

Methods Mol Biol. 2021:2280:189-198. doi: 10.1007/978-1-0716-1286-6_12.

Abstract

To perform their action usually flavoproteins interact transiently with a variety of molecular partners, whose binding is reciprocally affected and often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe an approach for the quantitative characterization of the redox-controlled interaction of the mammalian apoptosis inducing factor (AIF) with one of its known protein partners, namely, the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). In particular, we report a protocol for the titration of the flavoprotein in both in its oxidized and reduced states with CHCHD4, using an implementation of the MicroScale Thermophoresis (MST) technique.

Keywords: Microscale thermophoresis; Protein–ligand interaction; Protein–protein interaction; Redox-linked binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation
  • Anaerobiosis
  • Animals
  • Apoptosis Inducing Factor / chemistry*
  • Apoptosis Inducing Factor / genetics
  • Apoptosis Inducing Factor / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / growth & development*
  • Fluorescence
  • Mice
  • Mitochondrial Membrane Transport Proteins / metabolism*
  • Mitochondrial Precursor Protein Import Complex Proteins
  • Oxidation-Reduction
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spectrophotometry

Substances

  • Apoptosis Inducing Factor
  • Chchd4 protein, mouse
  • Mitochondrial Membrane Transport Proteins
  • Mitochondrial Precursor Protein Import Complex Proteins
  • AIFM1 protein, mouse
  • Recombinant Proteins