How fluorescent labelling alters the solution behaviour of proteins

Abstract

A complete understanding of the role of molecular anisotropy in directing the self assembly of colloids and proteins remains a challenge for soft matter science and biophysics. For proteins in particular, the complexity of the surface at a molecular level poses a challenge for any theoretical and numerical description. A soft matter approach, based on patchy models, has been useful in describing protein phase behaviour. In this work we examine how chemical modification of the protein surface, by addition of a fluorophore, affects the physical properties of protein solutions. By using a carefully controlled experimental protein model (human gamma-D crystallin) and numerical simulations, we demonstrate that protein solution behaviour defined by anisotropic surface effects can be captured by a custom patchy particle model. In particular, the chemical modification is found to be equivalent to the addition of a large hydrophobic surface patch with a large attractive potential energy well, which produces a significant increase in the temperature at which liquid-liquid phase separation occurs, even for very low fractions of fluorescently labelled proteins. These results are therefore directly relevant to all applications based on the use of fluorescent labelling by chemical modification, which have become increasingly important in the understanding of biological processes and biophysical interactions.

Limiting the valence: advancements and new perspectives on patchy colloids, soft functionalized nanoparticles and biomolecules

“…121 Consequently, the characterization of the phase behavior of protein solutions is mainly based on interactions with visible light that can be measured with common laboratory equipment and that provide insight into average properties. 21,[122][123][124] A recent review of the physics of protein self-assembly in the bulk is given in ref.…”

Model for screened, charge-regulated electrostatics of an eye lens protein: Bovine gammaB-crystallin

“…Because many known cataractogenic mutations of γ -crystallins involve changes of residue charge, it is natural to study the protonation configuration probability distributions in detail. While many models of orientation-dependent protein-protein interactions have been developed at various levels of coarse graining [3–6,11,132–134], some of which incorporate charge regulation, including models for lysozyme interactions [4–6,132,135,136] and for gamma crystallin interactions [52,88,137], achieving the degree of fine graining for the more predictive modeling needed in many contexts remains an outstanding challenge [19]. …”

Heteroprotein complex coacervation: A generic process

“…Heteroprotein complex coacervation is fully reversible when the balance of attractive and repulsive forces deviates from optimum conditions that constitute an essential property with delivery system perspective in mind. Constrained structural feature and surface charge anisotropy (surface charge density, size and shape of the patches) orient the interactions to specific domains on the surface of the proteins [14,15]. This constitutes specificities compared to complex coacervation involving polyelectrolytes and explain for a part that the (+/-) stoichiometry is not a sufficient condition for heteroprotein coacervation.…”