Biology subjects

GrandPre, T.

Publications and source records attributed to GrandPre, T..

3 recordsLinked to original sources

bioRxiv2023

Effects of linker length on phase separation: lessons from the Rubisco-EPYC1 system of the algal pyrenoid

Biomolecular condensates are membraneless organelles formed via phase separation of macromolecules, typically consisting of bond-forming "stickers" connected by flexible "linkers". Linkers have diverse roles, such as occupying space and facilitating interactions. To understand how linker length relative to other lengths affects condensation, we focus on the pyrenoid, which enhances photosynthesis in green algae. Specifically, we apply coarse-grained simulations and analytical theory to the pyrenoid proteins of Chlamydomonas reinhardtii: the rigid holoenzyme Rubisco and its flexible partner EPYC1. Remarkably, halving EPYC1 linker lengths decreases critical concentrations by ten-fold. We attribute this difference to the molecular "fit" between EPYC1 and Rubisco. Varying Rubisco sticker locations reveals that the native sites yield the poorest fit, thus optimizing phase separation. Surprisingly, shorter linkers mediate a transition to a gas of rods as Rubisco stickers approach the poles. These findings illustrate how intrinsically disordered proteins affect phase separation through the interplay of molecular length scales.

biophysics↗

bioRxiv2022

Phase-separating pyrenoid proteins form complexes in the dilute phase

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play a significant role in the stability, dynamics, and regulation of condensates.

biophysics↗

bioRxiv2021

Kinetic frustration by limited bond availability controls the LAT protein condensation phase transition on membranes

LAT is a membrane-linked scaffold protein that undergoes a phase transition to form a two-dimensional protein condensate on the membrane during T cell activation. Governed by tyrosine phosphorylation, LAT recruits various proteins that ultimately enable condensation through a percolation network of discrete and selective protein-protein interactions. Here we describe detailed kinetic measurements of the phase transition, along with coarse-grained model simulations, that reveal LAT condensation is kinetically frustrated by the availability of bonds to form the network. Unlike typical miscibility transitions in which compact domains may coexist at equilibrium, the LAT condensates are dynamically arrested in extended states, kinetically trapped out of equilibrium. Modeling identifies the structural basis for this kinetic arrest as the formation of spindle arrangements, favored by limited multivalent binding interactions along the flexible, intrinsically disordered LAT protein. These results reveal how local factors controlling the kinetics of LAT condensation enable formation of different, stable condensates, which may ultimately coexist within the cell.

biophysics↗