Unraveling the Physicochemical Determinants of Protein Liquid-liquid Phase Separation by Nanoscale Infrared Vibrational Spectroscopy

Francesco S Ruggeri; Alyssa M Miller; Michele Vendruscolo; Tuomas P J Knowles

doi:10.21769/BioProtoc.4122

Unraveling the Physicochemical Determinants of Protein Liquid-liquid Phase Separation by Nanoscale Infrared Vibrational Spectroscopy

Bio Protoc. 2021 Aug 20;11(16):e4122. doi: 10.21769/BioProtoc.4122.

Authors

Francesco S Ruggeri^{1

2}, Alyssa M Miller¹, Michele Vendruscolo¹, Tuomas P J Knowles^{1

3}

Affiliations

¹ Laboratories of Organic and Physical Chemistry, Stippeneng 4, 6703 WE, Wageningen University and Research, The Netherlands.
² Department of Chemistry, University of Cambridge, Cambridge, UK.
³ Cavendish Laboratory, University of Cambridge, Cambridge, UK.

Abstract

The phenomenon of reversible liquid-liquid phase separation of proteins underlies the formation of membraneless organelles, which are crucial for cellular processes such as signalling and transport. In addition, it is also of great interest to uncover the mechanisms of further irreversible maturation of the functional dense liquid phase into aberrant insoluble assemblies due to its implication in human disease. Recent advances in methods based on atomic force microscopy (AFM) have made it possible to study protein condensates at the nanometer level, providing unprecedented information on the nature of the intermolecular interactions governing phase separation. Here, we provide an in-depth description of a protocol for the characterisation of the morphology, stiffness, and chemical properties of protein condensates using infrared nanospectroscopy (AFM-IR).

Keywords: Atomic force microscopy; Infrared nanospectroscopy; Liquid-liquid phase separation; Single-molecule biophysics.