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

## TL;DR

This paper introduces a new method to control protein phase separation using NMR and targeted mutations.

## Contribution

The novel protocol integrates LLPS assays, NMR spectroscopy, and mutagenesis to regulate protein phase separation at the residue level.

## Key findings

- The workflow links residue-specific structural features to macroscopic LLPS behavior.
- Targeted amino acid substitutions can suppress or enhance phase separation.
- The protocol is demonstrated using the VAPB MSP domain as an example.

## 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.