Single-molecule digital sizing of proteins in solution

Georg Krainer (), Raphael P. B. Jacquat, Matthias M. Schneider, Timothy J. Welsh, Jieyuan Fan, Quentin A. E. Peter, Ewa A. Andrzejewska, Greta Šneiderienė, Magdalena A. Czekalska, Hannes Ausserwoeger, Lin Chai, William E. Arter, Kadi L. Saar, Therese W. Herling, Titus M. Franzmann, Vasilis Kosmoliaptsis, Simon Alberti, F. Ulrich Hartl, Steven F. Lee and Tuomas P. J. Knowles ()
Additional contact information
Georg Krainer: University of Graz
Raphael P. B. Jacquat: University of Cambridge
Matthias M. Schneider: University of Cambridge
Timothy J. Welsh: University of Cambridge
Jieyuan Fan: University of Cambridge
Quentin A. E. Peter: University of Cambridge
Ewa A. Andrzejewska: University of Cambridge
Greta Šneiderienė: University of Cambridge
Magdalena A. Czekalska: University of Cambridge
Hannes Ausserwoeger: University of Cambridge
Lin Chai: University of Cambridge
William E. Arter: University of Cambridge
Kadi L. Saar: University of Cambridge
Therese W. Herling: University of Cambridge
Titus M. Franzmann: Technische Universität Dresden
Vasilis Kosmoliaptsis: University of Cambridge, Addenbrooke’s Hospital
Simon Alberti: Technische Universität Dresden
F. Ulrich Hartl: Max-Planck Institute of Biochemistry
Steven F. Lee: University of Cambridge
Tuomas P. J. Knowles: University of Cambridge

Nature Communications, 2024, vol. 15, issue 1, 1-19

Abstract: Abstract The physical characterization of proteins in terms of their sizes, interactions, and assembly states is key to understanding their biological function and dysfunction. However, this has remained a difficult task because proteins are often highly polydisperse and present as multicomponent mixtures. Here, we address this challenge by introducing single-molecule microfluidic diffusional sizing (smMDS). This approach measures the hydrodynamic radius of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. smMDS allows for ultrasensitive sizing of proteins down to femtomolar concentrations and enables affinity profiling of protein interactions at the single-molecule level. We show that smMDS is effective in resolving the assembly states of protein oligomers and in characterizing the size of protein species within complex mixtures, including fibrillar protein aggregates and nanoscale condensate clusters. Overall, smMDS is a highly sensitive method for the analysis of proteins in solution, with wide-ranging applications in drug discovery, diagnostics, and nanobiotechnology.

Date: 2024
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DOI: 10.1038/s41467-024-50825-9

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