Ultrafast Spectroscopy Reveals Slow Water Dynamics in Biocondensates

Cells achieve high spatiotemporal control over biochemical processes through compartmentalization to membrane-bound, as well as membraneless organelles that assemble by liquid-liquid phase separation. Characterizing the balance of forces within these environments is essential to understanding their stability and function, and water is an integral part of the condensate, playing a nontrivial role in mediating the electrostatic and hydrogen bonding interactions. Here we investigate the picosecond hydrogen-bond dynamics of a model biocondensate consisting of a peptide Poly-L-Arginine and the nucleic acid adenosine monophosphate (AMP) using ultrafast two-dimensional infrared (2D IR) spectroscopy. We investigate three vibrational modes, the arginine side chain C=N stretches, an AMP ring mode, and the amide backbone carbonyl stretches, to provide different perspectives. In general, dynamics slow down considerably between the dilute phase and the condensate phase for each vibrational probe. For example, the arginine side chain C=N modes slows from 0.38 ps to 2.26 ps due to strong electrostatic interactions. Hydrogen bond lifetime computed from all-atom molecular dynamics simulations provide an atomistic interpretation. Simulations predict that a significant fraction of water molecules are highly constrained within the condensate. We attribute this slowdown in dynamics to a highly disordered and extremely crowded water environment.