Work

A Protocol for Regulating Protein Liquid–Liquid Phase Separation Using NMR-Guided Mutagenesis

Year: 2026

Type: article

Abstract: Liquid-liquid phase separation (LLPS) underlies the formation of membraneless cellular compartments, yet experimental strategies that directly connect quantitative LLPS behavior with residue-level structural information remain limited. Here, we present an integrated protocol that combines quantitative LLPS assays with nuclear magnetic resonance (NMR) spectroscopy and structure-guided mutagenesis to regulate protein phase separation. Using the VAPB MSP domain as a representative example, this workflow links residue-specific structural features to macroscopic LLPS behavior and enables suppression or enhancement of phase separation through targeted amino acid substitutions. This protocol provides a generalizable framework for systematic, residue-level regulation of protein LLPS.

Source: Methods and Protocols

DOI: 10.3390/mps9010026

Authors: Mayu Enomoto-Kusano, Kyoko Furuita, Takashi S. Kodama, Chojiro Kojima

Institutions: Yokohama National University, Kyoto University, Kyoto University Institute for Chemical Research, The University of Osaka

Language: English

Topic: RNA Research and Splicing

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $2,172

Funders: Japan Agency for Medical Research and Development, Institute for Protein Research, Osaka University