Direct Observation of Adsorption Sites of Protein Impurities and Their Effects on Step Advancement of Protein Crystals

Es, Van Driessehe Alexander; Sazaki, Gen; 戴国亮,; Otálora, Fermı́n; Gavira, José A.; Matsui, Toshihiro; Yoshizaki, Izumi; Tsukamoto, Katsuo; Nakajima, Kazuo

doi:10.1021/cg8006684

Cited by 36 publications

(63 citation statements)

References 55 publications

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“…In virtually all cases, these effects are strongly supersaturation dependent. The predominant interpretation is that the crystal undergoes a self-purifying transition by moving from an impurity-saturated state at low supersaturation toward an effective state of impurity repulsion at high driving forces (42,43). This can be understood by realizing that the impurity surface concentration at a given supersaturation depends on the ratio of the characteristic time of impurity adsorption and the exposure time of the impurity binding sites on the surface (44).…”

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.

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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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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.

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165

204

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“…We have previously directly visualized elementary steps of protein crystals (>several nm in height) in aqueous protein solutions (∼1% reflectivity) with a sufficient contrast level by this technique (34)(35)(36)(37). Using LCM-DIM, we could also visualize dislocations in protein crystals during growth (38).…”

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confidence: 99%

Elementary steps at the surface of ice crystals visualized by advanced optical microscopy

Sazaki

Zepeda

Nakatsubo

et al. 2010

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

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150

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Due to the abundance of ice on earth, the phase transition of ice plays crucially important roles in various phenomena in nature. Hence, the molecular-level understanding of ice crystal surfaces holds the key to unlocking the secrets of a number of fields. In this study we demonstrate, by laser confocal microscopy combined with differential interference contrast microscopy, that elementary steps (the growing ends of ubiquitous molecular layers with the minimum height) of ice crystals and their dynamic behavior can be visualized directly at air-ice interfaces. We observed the appearance and lateral growth of two-dimensional islands on ice crystal surfaces. When the steps of neighboring two-dimensional islands coalesced, the contrast of the steps always disappeared completely. We were able to discount the occurrence of steps too small to detect directly because we never observed the associated phenomena that would indicate their presence. In addition, classical two-dimensional nucleation theory does not support the appearance of multilayered two-dimensional islands. Hence, we concluded that two-dimensional islands with elementary height (0.37 and 0.39 nm on basal and prism faces, respectively) were visualized by our optical microscopy. On basal and prism faces, we also observed the spiral growth steps generated by screw dislocations. The distance between adjacent spiral steps on a prism face was about 1∕20 of that on a basal face. Hence, the step ledge energy of a prism face was 1∕20 of that on a basal face, in accord with the known lowertemperature roughening transition of the prism face.in situ observation | monomolecular steps | two-dimensional nucleation growth | spiral growth I ce is one of the most abundant materials on earth, and its phase transition governs a wide variety of phenomena, such as weather, environment-related issues, life in a cryosphere, and cosmic evolution, etc. Hence the molecular-level understanding of ice crystal surfaces is crucially important. For example, ice crystal surfaces play a key role in heterogeneous physical/chemical reactions, such as the degradation of ozone and organic compounds adsorbed on ice crystal surfaces by UV light irradiation (1-4), the suppression of the growth of ice in living things by antifreeze proteins adsorbed on ice crystal surfaces (5-8), etc., as well as in the growth and sublimation/melting of ice crystals.A crystal bounded by flat crystal faces grows layer by layer (9, 10), utilizing laterally growing molecular layers that have the minimum height determined by the crystal structure. Hence, growing ends of such molecular layers, so-called "elementary steps," which ubiquitously exist on a crystal surface, play a key role during the physical/chemical reactions and the growth and sublimation/melting of ice crystals. Therefore to clarify such phenomena at the molecular level, first one has to observe elementary steps on ice crystal surfaces.Many optical microscopy studies have been carried out to date to observe the surface morphology of ice crystals, such ...

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“…Van Dreassche et al also revealed the relation between adsorption sites of impurities and their effects on step advancement by TIRF microscopy. 10 To our knowledge, we performed the first direct visualization of the attachment/detachment processes of fluorescent-labeled HEWL (F-HEWL) molecules on a relatively large interface (50 × 50 μm 2 ) between a tetragonal HEWL crystal (several hundred micrometers in thickness) and an equilibrium HEWL solution. 11 We revealed an induction period (∼70 min) after which the number density of F-HEWL molecules adsorbed mainly on steps increased linearly with the adsorption time.…”

Section: Introductionmentioning

confidence: 99%

Attachment and Detachment Processes of Individual Lysozyme Molecules on a Surface of a Monoclinic Lysozyme Crystal Studied by Fluorescent Single-Molecule Visualization

Dai

Zheng

Sazaki

et al. 2014

Crystal Growth & Design

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On a {101̅ } face of a monoclinic crystal of hen egg-white lysozyme (HEWL), we visualized the attachment and detachment processes of individual fluorescent-labeled HEWL (F-HEWL) molecules by a fluorescent single-molecule visualization technique. We measured the changes in number density of F-HEWL molecules, whose positions were not changed for longer than a certain residence time, as a function of an adsorption time. We first confirmed that under an equilibrium condition, there was an "induction period" (∼120 min) of the attachment/detachment processes, during which period the number density remained constant. After the induction period, the number density increased linearly with the adsorption time, as it was recently found on a tetragonal HEWL crystal [Dai, G. L. Cryst. Growth Des 2011, 11(1), 88−92]. In addition, we performed similar measurements under a supersaturated condition. Then we found that supersaturation significantly enhanced the attachment process after the induction time. The attachment/detachment processes finally reached a steady state, in which the attachment rate was higher than the detachment one. Moreover, we also found that in a rare case, an F-HEWL molecule adsorbed on a step laterally moved following the advancement of a growing step.

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Direct Observation of Adsorption Sites of Protein Impurities and Their Effects on Step Advancement of Protein Crystals

Cited by 36 publications

References 55 publications

Role of clusters in nonclassical nucleation and growth of protein crystals

Role of clusters in nonclassical nucleation and growth of protein crystals

Elementary steps at the surface of ice crystals visualized by advanced optical microscopy

Attachment and Detachment Processes of Individual Lysozyme Molecules on a Surface of a Monoclinic Lysozyme Crystal Studied by Fluorescent Single-Molecule Visualization

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