Mesoscopic Protein Rich Phases in Ribonuclease a Solution



Journal Title

Journal ISSN

Volume Title



Dense liquid phases called clusters exist in protein solutions and act as a precursor to crystal nucleation. Clusters have a narrow size distribution and submicron size. These clusters are present in a minor fraction of total protein, not affecting the properties of the solution, but are essential precursors for the nucleation of ordered species. The experiments here are done with acidic protein solution of Ribonuclease A (RNase A). Here we studied the effect of ionic strength, urea and temperature with the help of Brownian microscopy (BM), static light scattering (SLS), and several biochemical assays. The cluster size distribution was measured for three days with BM and revealed a consistently narrow size distribution through this time period, indicating stability of clusters. The clusters respond to the change in protein concentration, this change is exhibited as decrease in cluster volume fraction as the protein concentration was increased, indicating these clusters are not irreversible aggregates. The intermolecular forces were varied by changing the ionic strength with NaCl, (NH4)2SO4 and CH3COONa and quantified by measuring second virial coefficient by SLS. The data revealed that intermolecular repulsion decreases the cluster size and cluster volume fraction. The hydrophobic interactions between the exposed hydrophobic residues due to partial unfolding and solvent was studied by the addition of urea, a chaotropic agent that destroys the shell of structured water around protein molecules. Elman’s assay was used to quantify the free thiols and the degree of unfolding was studied using ANS and ThT assays. The unfolding of protein by breaking disulphide bonds by heating showed an increase in cluster size and cluster volume fraction indicating the role of protein unfolding to aid the cluster formation. These experiments demonstrated the relevance of partial unfolding and intermolecular interactions in formation of clusters.



Proteins, Ribonuclease A, Clusters, Light scattering, Assays