Step velocity in tetragonal lysozyme growth as a function of impurity concentration and mass transport conditions

Dold, P.; Ono, E.; Tsukamoto, Katsuo; Sazaki, Gen

doi:10.1016/j.jcrysgro.2006.04.116

Cited by 41 publications

(54 citation statements)

References 49 publications

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“…We observed orthorhombic lysozyme crystals using phase contrast microscopy (PCM), and we measured their growth rates for comparison with those observed using TEM. For these measurements, we used a growth cell (32) in which a seed crystal and the growth solution were sealed. Seed crystals for the PCM observations were produced using a crystallization solution (100-120 mg·mL −1 lysozyme plus 25 mg·mL −1 NaCl in the buffer solution).…”

Section: Methodsmentioning

confidence: 99%

Two types of amorphous protein particles facilitate crystal nucleation

Yamazaki

Kimura

Vekilov

et al. 2017

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

163

165

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Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used timeresolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.nucleation | protein | lysozyme | transmission electron microscopy | in situ observation C rystallization can be divided into two processes: nucleation and crystal growth. The crystal growth process has been well examined for a long time, yet the nucleation process is not understood; for example, the nucleation rate of crystals provides a textbook example of order-of-magnitude discrepancies between theoretical predictions and experimental results. Recent proposals have attributed these discrepancies to a nonclassical nucleation pathway, along which a structured crystalline embryo forms within a highly concentrated disordered precursor (1). This mechanism was first proposed for protein crystals (2, 3). Direct observations have demonstrated its applicability to organic (4), inorganic (5, 6), and colloidal (7) crystals. In proteins, clusters of protein molecules have been suggested as precursors; these clusters have mesoscopic sizes from several tens to several hundreds of nanometers and are considered to behave like liquids. It has also been suggested that the precursor is thermodynamically stable with respect to the mother liquid phase but is metastable or unstable with respect to the crystalline phase. The latter nature of the precursor differs from the stable macroscopically dense liquid formed as a result of the liquid-liquid phase separation (8). Such protein-rich mesoscopic clusters have been observed for many proteins, primarily using optical techniques, and have been tentatively identified as precursors for crystal nucleation (9-12). Several important questions concerning this mechanism remain unanswered. First, are the observed mesoscopic clusters actually liquid-like or solid-amorphous? Second, do they play an active role in crystal nucleation? In addition, finally, do the clusters serve as classical heteroge...

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Section: Methodsmentioning

confidence: 99%

Two types of amorphous protein particles facilitate crystal nucleation

Yamazaki

Kimura

Vekilov

et al. 2017

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

163

165

View full text Add to dashboard Cite

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“…To understand the possible factors affecting the quality of protein crystals, it is indispensable to clarify the effects of impurities, since impurities can significantly affect the growth kinetics, [1][2][3][4][5][6][7][8][9][10][11] morphology, [12][13][14][15] lattice disorder, 16 and hence quality of protein crystals. [17][18][19][20][21][22][23][24] Generally, impurities are considered to exhibit their effects on the growth process after they adsorb on a crystal surface.…”

Section: Introductionmentioning

confidence: 99%

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

Sazaki

戴国亮

et al. 2009

Crystal Growth & Design

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ABSTRACT:We measured noninvasively step velocities of elementary two-dimensional (2D) islands on {110} faces of tetragonal lysozyme crystals, under various supersaturations, by laser confocal microscopy combined with differential interference contrast microscopy. We studied the correlation between the effects of protein impurities on the growth of elementary steps and their adsorption sites on a crystal surface, using three kinds of proteins: fluorescent-labeled lysozyme (F-lysozyme), covalently bonded dimers of lysozyme (dimer), and a 18 kDa polypeptide (18 kDa). These three protein impurities suppressed the advancement of the steps. However, they exhibited different supersaturation dependencies of the suppression of the step velocities. To clarify the cause of this difference, we observed in situ the adsorption sites of individual molecules of F-lysozyme and fluorescent-labeled dimer (F-dimer) on the crystal surface by single-molecule visualization. We found that F-lysozyme adsorbed preferentially on steps (i.e., kinks), whereas F-dimer adsorbed randomly on terraces. Taking into account the different adsorption sites of F-lysozyme and F-dimer, we could successfully explain the different effects of the impurities on the step velocities. These observations strongly suggest that 18 kDa also adsorbs randomly on terraces. Seikagaku lysozyme exhibited a complex effect that could not alone be explained by the two major impurities (dimer and 18 kDa) present in Seikagaku lysozyme, indicating that trace amounts of other impurities significantly affect the step advancement.

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“…Lorber et al [31] correlated protein impurities (ovalbumin, BSA) in lysozyme solutions with changes in the total number and defect density of lysozyme crystals. Protein impurities have also been implicated in changes to growth rates on the 101 facet of tetragonal lysozyme crystals [12,34,50], the density of optical defects in lysozyme crystals [6] and limitations of X-ray diffraction resolution [7,33,43]. Even in the absence of contaminating impurities, structural micro-heterogeneities of lysozyme monomers have been linked to altered crystal habit and crystal quality [16].…”

Section: Introductionmentioning

confidence: 99%