Protein oligomerization in homogenous protein solutions Crosslinker:GlutaraldehydeOHC-CH 2 -CH 2 -CH 2 -CHO Y. Wang and O. Annunziata Langmuir, 24, (2008) Examples: lysozyme, Albumin

Effect of Protein oligomerization on LLPS temperature LLPS temperature increases as the degree of oligomerization increases, leading to the formation of crosslinked microdroplets at room temperature LLPS of protein monomer occurring at -10 °C Protein oligomerization performed at 20 °C

Effect of crosslinker concentration on the size of protein nucleating clusters. Radius of nucleating protein nanoclusters inversely proportional to crosslinker concentration (Dynamic light scattering)

Oligomerization-induced formation of protein microspheres Protein Concentration Temperature LLPS One Phase sample oligomerization

Oligomerization-induced formation of protein microspheres Protein Concentration Temperature sample One Phase LLPS oligomerization

Oligomerization-induced formation of protein microspheres The presence of the LLPS boundary affects the morphology of protein condensed phase. Protein Concentration Temperature sample One Phase LLPS oligomerization LLPS Y. Wang and O. Annunziata Langmuir, 24, (2008)

Scheme of a diffusion process in the presence of biopolymer gradient and uniform salt concentration. Diffusion of salt ions is described by D 21. MACOMOLECULE – SALT COUPLED DIFFUSION Annunziata et al. J. Phys. Chem. B, 110, (2006).

Rayleigh Interferometric Method The interferometric pattern yields the Refractive Index Profile Miller and Albright, Measurement of the Transport Properties of Fluids: Experimental Thermodynamics, p. 272 (1991). Annunziata et al., Langmuir, p (2005).

Annunziata et al. J. Am. Chem. Soc., 130, (2008).

Conclusions LLPS of protein solutions can be induced using polymers. Protein oligomerization enhances LLPS, provided the LLPS of the protein monomer exists. The outcome of protein oligomerization depends on the LLPS boundary of the protein monomer. Radius of nucleating protein nanoclusters is inversely proportional to the crosslinker concentration. Ternary diffusion can be used to characterize charge and solvation of macromolecules in solution. We have applied this method to a charged enzyme (lysozyme) and to a polymer (PEG). Comparison of solubility data with preferential-interaction coefficients allow us to demonstrate that the protein chemical potential in the crystalline phase strongly depends on salt concentration.