title: Do protein crystals nucleate within dense liquid clusters?
abstract: Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10(-3) of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in lysozyme and glucose isomerase solutions are locations for crystal nucleation.
authors.affiliation: Structural Biology Brussels (SBB), Vrije Universiteit Brussel, 1050 Brussels, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Maes
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX 77204, USA.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Vorontsova
authors.affiliation: Dipartimento de Fisica, Universita di Milano, 20133 Milano, Italy.
authors.forename: Marco A C
authors.initials: MA
authors.lastname: Potenza
authors.affiliation: Dipartimento de Fisica, Universita di Milano, 20133 Milano, Italy.
authors.forename: Tiziano
authors.initials: T
authors.lastname: Sanvito
authors.affiliation: Structural Biology Brussels (SBB), Vrije Universiteit Brussel, 1050 Brussels, Belgium.
authors.forename: Mike
authors.initials: M
authors.lastname: Sleutel
authors.affiliation: Dipartimento de Fisica, Universita di Milano, 20133 Milano, Italy.
authors.forename: Marzio
authors.initials: M
authors.lastname: Giglio
authors.affiliation: Structural Biology Brussels (SBB), Vrije Universiteit Brussel, 1050 Brussels, Belgium.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Vekilov
mesh_terms: D000818:Animals; D002645:Chickens; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D000067493:Dynamic Light Scattering / Q000379:methods; D050866:Liquid Crystals / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*
chemical_list: D009113:Muramidase
keywords: crystallization; nucleation; protein-rich clusters; two-step mechanism