Protein crystallization and phase diagrams

Asherie, Neer

doi:10.1016/j.ymeth.2004.03.028

Cited by 250 publications

(227 citation statements)

References 35 publications

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“…As a result, further growth stops, leading to an apparent solubility that is higher than the equilibrium value. 46 This may be why our solubility results for Sigma thaumatin with L-tartrate (solid squares in Figure 6B) are lower than those of the other investigators who measured the solubility upon growth.…”

Section: Sigma Thaumatincontrasting

confidence: 60%

“…To check that our results were selfconsistent, some solubility data was collected starting with supersaturated protein solutions, as well as undersaturated ones. 46 For the solubility measurements, the concentration of protein could not be determined accurately by direct UV extinction spectroscopy at 280 nm, as the tartrate ions also absorb around 280 nm. Instead, SE-HPLC was performed on each aliquot and the concentration of the protein was determined from the area of the well-resolved protein peak.…”

Section: Size-exclusion and Cation-exchange Chromatographymentioning

confidence: 99%

“…Liquid-liquid phase separation experiments show that this pigment associates with the protein. 46 Crude Natex thaumatin is also colored: it forms a dark-pink solution. The solutions of the purified protein, however, are colorless.…”

Section: Source Of Proteinmentioning

confidence: 99%

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Effects of Protein Purity and Precipitant Stereochemistry on the Crystallization of Thaumatin

Asherie

Ginsberg

Greenbaum

et al. 2008

Crystal Growth & Design

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ABSTRACT:Thaumatin is frequently used as a model protein in crystallization studies because it rapidly forms crystals in the presence of tartrate ions. The thermodynamic and kinetic properties of thaumatin crystals have been studied for almost 10 years, and the results are contradictory. Here we show that by using a homogeneous preparation of thaumatin and controlling the stereochemistry of the tartrate precipitant, it is possible to achieve consistent results for the protein solubility. To understand the role of protein impurities in the crystallization of thaumatin, we examined two commercial sources of the protein and characterized the heterogeneities therein. To examine the effect of precipitant stereochemistry, we crystallized thaumatin with L, D, and DL (racemic) tartrate ions. We suggest that the inconsistencies among previous results stem in part from the different behavior of thaumatin with the L and D enantiomers: the solubility of thaumatin crystals increases with temperature in L-tartrate, whereas it decreases with temperature in D-tartrate. Our results demonstrate the importance of using pure protein and stereochemically pure precipitants in the crystallization of proteins.

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Section: Sigma Thaumatincontrasting

confidence: 60%

Section: Size-exclusion and Cation-exchange Chromatographymentioning

confidence: 99%

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Effects of Protein Purity and Precipitant Stereochemistry on the Crystallization of Thaumatin

Asherie

Ginsberg

Greenbaum

et al. 2008

Crystal Growth & Design

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“…26,33,36,37 Liquid-liquid phase separation (LLPS) occurs for proteins interacting via short-ranged net attractive forces. 25,33,34 Indeed, the temperature at which LLPS occurs is sensitive to the strength of net attraction between proteins in solution and can be used to probe the relative changes in protein inter-particle interactions as a result of altered protein chemistry, changing solution conditions or the presence of a second protein type. 38,39 In this study, we experimentally assess the phase diagram for human gD-crystallin (HGD), which is a structural protein in the eye lens that undergoes LLPS due to short-ranged attractive interactions which dominate its behaviour at physiological pH (at the pI of the protein).…”

Section: Introductionmentioning

confidence: 99%

How fluorescent labelling alters the solution behaviour of proteins

Quinn

Gnan²,

James

et al. 2015

Phys. Chem. Chem. Phys.

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A complete understanding of the role of molecular anisotropy in directing the self assembly of colloids and proteins remains a challenge for soft matter science and biophysics. For proteins in particular, the complexity of the surface at a molecular level poses a challenge for any theoretical and numerical description. A soft matter approach, based on patchy models, has been useful in describing protein phase behaviour. In this work we examine how chemical modification of the protein surface, by addition of a fluorophore, affects the physical properties of protein solutions. By using a carefully controlled experimental protein model (human gamma-D crystallin) and numerical simulations, we demonstrate that protein solution behaviour defined by anisotropic surface effects can be captured by a custom patchy particle model. In particular, the chemical modification is found to be equivalent to the addition of a large hydrophobic surface patch with a large attractive potential energy well, which produces a significant increase in the temperature at which liquid-liquid phase separation occurs, even for very low fractions of fluorescently labelled proteins. These results are therefore directly relevant to all applications based on the use of fluorescent labelling by chemical modification, which have become increasingly important in the understanding of biological processes and biophysical interactions.

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“…Although this model is a crude approximation for the spatial description of the forces between proteins, it has been shown to provide a reasonably accurate description of the liquid-liquid phase diagram for globular proteins in solution. 19,20 For a fixed salt type, this model yields a semiquantitative description of the phase diagrams of these systems.…”

Section: Introductionmentioning

confidence: 99%

Liquid−Liquid Coexistence Surface for Lysozyme: Role of Salt Type and Salt Concentration

Wentzel

Gunton

2007

J. Phys. Chem. B

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The liquid-liquid phase separation curves for lysozyme in a salt solution are known to depend on salt type and salt concentration. For the case of monovalent cations, the cloud point temperature typically increases with increasing salt concentration, for fixed lysozyme concentration. For the case of divalent cations, however, a maximum in the cloud point temperature is observed that has been interpreted as being due to ion binding to the protein surface and subsequent water structuring. In this paper, we use a simple square well model due to Grigsby et al. (Biophys. Chem. 2001, 91, 231-243), whose well depth depends on salt type and salt concentration, to determine the phase coexistence surfaces from experimental data. The surfaces are shown as a function of temperature, salt concentration, and protein concentration for two typical salts, NaCl and MgCl 2 . These surfaces are calculated using the results of a single standard Monte Carlo simulation and a simple scaling argument and are in reasonably good agreement with known experimental results.

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Protein crystallization and phase diagrams

Cited by 250 publications

References 35 publications

Effects of Protein Purity and Precipitant Stereochemistry on the Crystallization of Thaumatin

Effects of Protein Purity and Precipitant Stereochemistry on the Crystallization of Thaumatin

How fluorescent labelling alters the solution behaviour of proteins

Liquid−Liquid Coexistence Surface for Lysozyme: Role of Salt Type and Salt Concentration

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