Biology subjects

Shiraki, K.

Publications and source records attributed to Shiraki, K..

4 recordsLinked to original sources

bioRxiv2026

Local aromatic interactions define temperature sensitivity of phase separation in an intrinsically disordered protein.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins is highly sensitive to environmental conditions, yet the molecular basis of sharp temperature responsiveness remains poorly understood. Here, we investigate a sequence-encoded mechanism underlying the pronounced temperature sensitivity of phase separation using Sup35NM, the intrinsically disordered domain of the yeast prion Sup35. We show that a tyrosine-rich local structural region within this domain encodes strong temperature responsiveness of droplet formation. Mutational analyses reveal that tyrosine residues mediate both intramolecular interactions that stabilize local structure and intermolecular interactions required for LLPS. Substitution of the tyrosine residues with alanine disrupts local structure and weakens intermolecular interactions, thereby diminishing temperature sensitivity. In contrast, substitution with phenylalanine promotes rapid droplet gelation, abolishes internal fluidity, suppresses amyloid formation, and confers resistance to temperature-induced dissolution. Based on these findings, we propose a molecular model in which finely tuned, moderately weak aromatic interactions among tyrosines enable reversible local compaction that is sensitive to temperature, thereby generating a sharp phase transition. These results suggest that amino acid sequences encode not only phase separation propensity but also the sensitivity of condensates to environmental perturbations, providing a framework for understanding how intrinsically disordered proteins act as molecular sensors of cellular conditions.

biochemistry↗

bioRxiv2024

Monovalent Ion Effect on Liquid-Liquid Phase Separation of Aqueous Polyphosphate-Salt Mixtures

Polyphosphate (polyP) is one of the most conserved biomacromolecules and can form aggregates, such as polyP granules in bacteria, which are generated through liquid-liquid phase separation (LLPS). Studies have examined the mechanism of polyP aggregation using LLPS systems containing artificial polyP molecules as aggregation system models, where LLPS is typically induced by multivalent salts and polyelectrolytes. Although the typical concentrations of monovalent ions in living cells are approximately 100 times higher than those of divalent ions, the effects of monovalent ions on the LLPS of polyP solutions are little known. This study demonstrated that submolar NaCl induces LLPS of polyP solutions, whereas other monovalent salts did not at the same concentrations. Small-angle X-ray scattering measurements revealed that NaCl significantly stabilizes the intermolecular association of polyP, inducing LLPS. These findings suggest that the modulation of monovalent ion concentrations is an underlying mechanism of polyP aggregate formation/deformation within living cells. TOC GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/592046v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@11bc9f4org.highwire.dtl.DTLVardef@167ee3corg.highwire.dtl.DTLVardef@49e055org.highwire.dtl.DTLVardef@27032a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Reentrant condensation of a multicomponent complex system of biomolecules induced by polyphosphate

Reentrant condensation (RC) is a phase behavior of protein solution comprising at least two components. In RC, a protein state varies from one phase to two phases and then back to one phase as the concentration of one component monotonically increases. To understand the phase behavior of multicomponent complex solutions of biomolecules, it is worth constructing an experimental multicomponent system that exhibits RC behavior. Here, we used a cola/milk mixture to investigate RC of a multicomponent complex system and explained the RC mechanism by reducing the system to two pure components, polyphosphate (polyP) and casein. In the multicomponent complex system, RC was observed with 20-60% cola and 1% milk. In the pure system, RC occurred with 0.01-2 mM tetraphosphate and 0.5 mg/ml casein. Moreover, the phase diagram showed that the condensation of casein depended on the chain length of the polyP. The present study succeeded in experimentally inducing RC in a multicomponent system and reproducing RC even when the system was reduced to its pure components. The fact that RC can be experimentally induced using common materials will provide important insights into the understanding of phase-separation behavior of biomolecules.

biochemistry↗

bioRxiv2020

Hyperactivation of L-lactate oxidase by liquid-liquid phase separation

Liquid droplets formed by liquid-liquid phase separation are attracting attention as functional states of proteins in living cells. Liquid droplets are thought to activate enzymatic reactions by assembling the required molecules. Thus, liquid droplets usually increase the affinity of an enzyme to its substrates, leading to decreased KM values. In this study, we demonstrate a new mechanism of enzyme activation in the droplets using Llactate oxidase (LOX). In the presence of poly-L-lysine (PLL), LOX formed droplets with diameters of hundreds of nanometers to tens of micrometers, stabilized by electro-static interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold decrease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increasing kcat by the formation of the liquid droplet. Interestingly, the conformation of LOX changed in the liquid droplet, probably leading to increased kcat value. Understanding enzyme activation in the droplets provides essential information about enzyme function in living cells in addition to biotechnology applications.

biochemistry↗