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

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

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Article in Methods and protocols, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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5 · Who and what money

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4 authors.

Mayu Enomoto-KusanoGraduate School of Engineering Science, Yokohama National University, Tokiwadai 79-5, Hodogaya-ku, Yokohama 240-8501, Kanagawa, Japan.

Kyoko FuruitaInstitute for Protein Research, The University of Osaka, 3-2 Yamadaoka, Suita 565-0871, Osaka, Japan.

Takashi S KodamaInstitute for Protein Research, The University of Osaka, 3-2 Yamadaoka, Suita 565-0871, Osaka, Japan.ORCID 0000-0001-6820-1907

Chojiro KojimaGraduate School of Engineering Science, Yokohama National University, Tokiwadai 79-5, Hodogaya-ku, Yokohama 240-8501, Kanagawa, Japan.ORCID 0000-0003-2723-8249

Funding

Institute for Protein Research, Osaka University CR-24-05Japan Agency for Medical Research and Development JP24ama121001Japan Society for the Promotion of Science JP22H05536, JP22K19184, JP23K27109, JP23K18030, JP25KJ0230, JPMJSP2178Ministry of Education, Culture, Sports, Science and Technology NMR Platform

6 · The paper itself

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.

Indexed as

liquid–liquid phase separationNMR-guided mutagenesisnuclear magnetic resonanceprotein condensatesprotein dynamicsVAPB

Identifiers

PMID41718328

DOI10.3390/mps9010026

PMCPMC12922142

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