Physicochemical Aspects of the Two-Step Mechanism of Nucleation in Protein Solutions



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Protein-rich liquid clusters exist in solutions of numerous proteins. They play the role of nucleation precursors of ordered solids of both folded proteins and partially misfolded chains. Examples include protein crystals, sickle-cell hemoglobin polymers, and amyloid fibrils. The clusters hold the key to the understanding and control of protein aggregation, and hence insights of their physical properties is needed for development of successful crystallization recipes. We prove that protein clusters are not the nuclei of the dense liquid but rather represent a new phase which exists in homogeneous field of the protein phase diagram. With nuclear magnetic resonance method we find the regions of protein molecules flexibility, potentially participating in cluster formation. We prove that water structuring interactions and partial protein unfolding contribute to clustering. We show that common organic additives used in crystallization increase cluster volume fraction and surface area. The tests of insulin protein solutions explain why the two-step mechanism of nucleation is selected. We develop a new spatial cross-correlation tracking method suitable for large (> λ/2) clusters. Monitoring of shape variations of intensity patterns of a single cluster indicates that protein clusters are liquid. We employ depolarized oblique illumination microscopy to study the nucleation process and we show that crystals of lysozyme and glucose isomerase proteins indeed nucleate within protein-rich liquid clusters. These are the first experiments of a direct observation of a two-step mechanism of nucleation in protein solutions.



Protein clusters, Two-step mechanism of nucleation


Portions of this document have appeared in: Vorontsova, Maria A., Ho Yin Chan, Vassiliy Lubchenko, and Peter G. Vekilov. "Lack of dependence of the sizes of the mesoscopic protein clusters on electrostatics." Biophysical journal 109, no. 9 (2015): 1959-1968. DOI: 10.1016/j.bpj.2015.09.025; and in: Li, Ye, Vassiliy Lubchenko, Maria A. Vorontsova, Luis Filobelo, and Peter G. Vekilov. "Ostwald-like ripening of the anomalous mesoscopic clusters in protein solutions." The Journal of Physical Chemistry B 116, no. 35 (2012): 10657-10664. DOI: 10.1021/jp303316s; and in: Vorontsova, Maria A., Dominique Maes, and Peter G. Vekilov. "Recent advances in the understanding of two-step nucleation of protein crystals." Faraday discussions 179 (2015): 27-40. DOI: 10.1039/C4FD00217B.