A Metastable Prerequisite for the Growth of Lumazine Synthase Crystals

Gliko, Olga; Neumaier, Nikolaus; Pan, Weichun; Haase, Ilka; Fischer, Markus; Bacher, Adelbert; Weinkauf, Sevil; Vekilov, Peter G.

doi:10.1021/ja043218k

Cited by 143 publications

(207 citation statements)

References 51 publications

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“…At the lowest temperature, the barriers encountered along the non-classical path are even lower so the advantage of this path is even greater. This picture agrees well with that developed by Vekilov and co-workers who have observed, by means of dynamic light-scattering, the presence of short-lived dense liquid droplets in protein solutions [6]. The results for the LJ system, shown in Fig.…”

supporting

confidence: 92%

“…More recently, the simple picture has also been challenged by novel experimental investigations. Vekilov and co-workers have shown that, prior to crystallization, protein solutions harbor metastable droplets of dense fluid and they have suggested that these droplets are necessary precursors of crystallization [4,5,6,7]. The picture that emerges is one of the formation of metastable droplets of dense fluid which then subsequently crystallize.…”

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Theoretical Evidence for a Dense Fluid Precursor to Crystallization

2006

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We present classical density functional theory calculations of the free energy landscape for fluids below their triple point as a function of density and crystallinity. We find that for both a model globular protein and for a simple atomic fluid modeled with a Lennard-Jones interaction, it is free-energetically easier to crystallize by passing through a metastable dense fluid in accord with the Ostwald rule of stages but in contrast to the alternative of ordering and densifying at once as assumed in the classical picture of crystallization.PACS numbers: 82.60. Nh,87.15.Nn,05.20.Jj Crystallization is an intricate process of fundamental importance in many areas of physics, chemistry and engineering. The classical picture of crystallization from supersaturated solutions goes back to Gibbs and consists of the spontaneous formation of crystalline clusters which then either grow or shrink depending on the relative importance of the free energy gain due to the lower bulk free energy of the crystal cluster and the free energy penalty due to the surface tension between the two phases. In this picture, the local density is the only order parameter: the crystalline cluster is (in general) denser than the fluid. In recent years, this picture has been called into question by simulation, theory and experiment for the particular and important case of the crystallization of globular proteins. ten Wolde and Frenkel (hereafter tWF) showed by means of simulation that the free energy landscape of protein crystal clusters as a function of the number of atoms in the cluster and the "crystallinity " favored paths leading from no clusters to clusters with low order to ordered clusters over paths moving from no clusters directly to ordered clusters [1]. This picture was confirmed by Talanquer and Oxtoby [2] and Shiryayev and Gunton[3] who showed using a parameterized van der Waals-type model of globular proteins that surface wetting did indeed lower the free energy of crystal clusters. More recently, the simple picture has also been challenged by novel experimental investigations. Vekilov and co-workers have shown that, prior to crystallization, protein solutions harbor metastable droplets of dense fluid and they have suggested that these droplets are necessary precursors of crystallization [4,5,6,7]. The picture that emerges is one of the formation of metastable droplets of dense fluid which then subsequently crystallize. In this paper, we show by means of classical density functional theory calculations that there is an intrinsic free-energy advantage in first densifying into a metastable densefluid state and then crystallizing rather than following the classical path which goes directly from gas to crystal. Furthermore, our calculations suggest that a similar advantage exists for fluids of small molecules, modeled here via the Lennard-Jones (LJ) interaction, thus indicating that this mechanism may underlie most crystallization processes.The starting point for our analysis is classical density functional theory (DFT) which is based...

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Theoretical Evidence for a Dense Fluid Precursor to Crystallization

2006

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“…Two different models have been postulated to account for the quasi-instantaneous formation of multilayer stacks: the microcrystal-sedimentation scenario (28,33) and the cluster-assimilation scenario (34,35). In brief, the former model assumes coincidental sedimentation of freshly bulk-nucleated microcrystals in an at-random orientation onto the macrocrystal, followed by a rapid reorientation to align with the underlying lattice leading to a flawless merging of both phases (36).…”

Section: Resultsmentioning

confidence: 99%

Role of clusters in nonclassical nucleation and growth of protein crystals

Sleutel

Driessche

2014

Proc. Natl. Acad. Sci. U.S.A.

165

204

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The development of multistep nucleation theory has spurred on experimentalists to find intermediate metastable states that are relevant to the solidification pathway of the molecule under interest. A great deal of studies focused on characterizing the so-called "precritical clusters" that may arise in the precipitation process. However, in macromolecular systems, the role that these clusters might play in the nucleation process and in the second stage of the precipitation process, i.e., growth, remains to a great extent unknown. Therefore, using biological macromolecules as a model system, we have studied the mesoscopic intermediate, the solid end state, and the relationship that exists between them. We present experimental evidence that these clusters are liquidlike and stable with respect to the parent liquid and metastable compared with the emerging crystalline phase. The presence of these clusters in the bulk liquid is associated with a nonclassical mechanism of crystal growth and can trigger a self-purifying cascade of impurity-poisoned crystal surfaces. These observations demonstrate that there exists a nontrivial connection between the growth of the macroscopic crystalline phase and the mesoscopic intermediate which should not be ignored. On the other hand, our experimental data also show that clusters existing in protein solutions can significantly increase the nucleation rate and therefore play a relevant role in the nucleation process.prenucleation clusters | phase transition | self-purification T he process of crystallization is generally considered to occur in two consecutive but very different stages: nucleation and growth. The first stage was already studied more than two centuries ago by Gibbs, who considered the nucleation of water droplets from a supersaturated vapor through the formation of globulae. He was the first to develop a thermodynamic formalism of nucleation by considering the generation of nuclei of a liquid phase as a density fluctuation of the parent phase (1, 2). Since then, Gibbs' nucleation theory has been extended to the nucleation of solid phases from solution and gaseous phases from which the current paradigm (based on the capillary approximation) for nucleation emerged, i.e., the classical nucleation theory ]. There is, however, an increasing body of evidence that shows that CNT can fail drastically when used in cases where the implicit and explicit assumptions of CNT are poorly justified (for a full dissection of the limitations, see refs. 9-11). An obvious situation where CNT will have limited applicability is in cases where the old and the new phases differ by at least two order parameters, e.g., density and structure (12).Recent theoretical, computational, and experimental efforts have demonstrated that densification and local increase in crystallinity need not occur simultaneously (13)(14)(15)(16)(17)(18)(19)(20). These results have inspired the development of a new approach that considers nucleation from solution as a multistep process attributing key roles to metastabl...

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“…The 2D clusters might be ordered or disordered, akin to a 2D liquid formed in the pool of hexamers adsorbed on the terraces: examples of liquid phases in 2D systems (50) have been discussed (51). A 3D analog of this process would be layer generation by the landing of dense liquid droplets on the surface of an existing crystal (52). In further analogy, the stacks of layers formed by 3D nucleation do not grow in height but expand laterally (52), a behavior similar to that of the mounds described above.…”

Section: The Mechanism Of Mound Formation: 2d Clusters On the Terracesmentioning

confidence: 99%

A fast response mechanism for insulin storage in crystals may involve kink generation by association of 2D clusters

Georgiou

Vekilov

2006

Proc. Natl. Acad. Sci. U.S.A.

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Crystals that are likely rhombohedral of Zn-insulin hexamers form in the islets of Langerhans in the pancreases of many mammals. The suggested functions of crystal formation is to protect the insulin from proteases and increase the degree of conversion of soluble proinsulin. To accomplish these ends, crystal growth should be fast and adaptable to rate fluctuations in the conversion reaction. Zn-insulin crystals grow layer by layer. Each layer spreads by the attachment of molecules to kinks located at the layers' edges, also called steps. The kinks are thought to be generated either by thermal fluctuations, as postulated by Gibbs, or by 1D nucleation of new crystalline rows. The kink density determines the rate at which steps advance, and these two kink-generation mechanisms lead to weak near-linear responses of the growth rate to concentration variations. We demonstrate for the crystallization of Zn-insulin a mechanism of kink generation whereby 2D clusters of several insulin molecules preformed on the terraces between steps associate to the steps. This mechanism results in severalfold-higher kink density, a faster rate of crystallization, and a high sensitivity of the kinetics to small increases of the solute concentration. If the found mechanism operates during insulin crystallization in vivo, it could be a part of the biological regulation of insulin production and function. For other crystallizing materials in biological and nonbiological systems, this mechanism provides an understanding of the often seen nonlinear acceleration of the kinetics.biological function ͉ crystallization mechanisms ͉ in situ atomic force microscopy ͉ steps

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A Metastable Prerequisite for the Growth of Lumazine Synthase Crystals

Cited by 143 publications

References 51 publications

Theoretical Evidence for a Dense Fluid Precursor to Crystallization

Theoretical Evidence for a Dense Fluid Precursor to Crystallization

Role of clusters in nonclassical nucleation and growth of protein crystals

A fast response mechanism for insulin storage in crystals may involve kink generation by association of 2D clusters

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