Dynamic light scattering analysis of solutions from which lysozyme crystals grow

Bishop, John Bradford; Fredericks, William J.; Howard, Sandra B.; Sawada, T.

doi:10.1016/0022-0248(92)90224-7

Cited by 20 publications

(10 citation statements)

References 22 publications

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“…The decrease of the diffusion coefficient with time at constant protein concentration prior to nucleation (Fig. 6) has already been observed and explained by Bishop (Bishop, Fredericks, Howard & Sawada, 1992). It is due to strong molecular interactions leading to crystallization.…”

Section: Discussionmentioning

confidence: 72%

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Influence of polydispersity on protein crystallization: a quasi-elastic light-scattering study applied to α-amylase

Veesler

Marcq²,

Lafont³

et al. 1994

Acta Cryst D

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The early stages of the crystallization process of porcine pancreatic a-amylase were investigated by quasi-elastic light scattering. It is shown that at 288 and 293 K the diffusion coefficient does not monotonically change with increasing protein concentration but passes through a maximum at 10 mg ml-t In supersaturated solutions, prior to nucleation, the protein is strictly monodisperse. Nucleation induces the formation of aggregates and a polydispersity of, for example, 18% for an initial supersaturation C/C,. = 5.8. Monodispersity is restored after the nuclei have grown and partially consumed the solute. On the other hand, polydispersity increases up to 20% at 298 K if the protein concentration decreases to 3-4 mg ml-i, values at which the solutions are undersaturated. When the protein concentration exceeds 5-6 mg ml-~ the protein becomes monodisperse again. These results, confirmed by those of another system we are studying (bovine pancreatic trypsin inhibitor), are at variance with the statements that supersaturation is always at the origin of aggregation and polydispersity, and that in undersaturated solutions the diffusion coefficient should remain constant for obtaining crystals once the solutions are supersaturated.

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Section: Discussionmentioning

confidence: 72%

“…As previously suggested (Bishop, Fredericks, Howard & Sawada, 1992), attractive interactions are detectable only above some threshold concentration corresponding to an intermolecular distance of 150 A for lysozyme, a value to be compared with 240 A in the case of a-amylase for the concentration of 10 mg ml-…”

Section: Discussionmentioning

confidence: 80%

Influence of polydispersity on protein crystallization: a quasi-elastic light-scattering study applied to α-amylase

Veesler

Marcq²,

Lafont³

et al. 1994

Acta Cryst D

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“…It was, therefore, necessary to determine which substances are retained by the membrane. Because low ionic strength acidic solutions of lysozyme have been shown to be monomeric by light scattering (Bishop et al, 1992) and dialysis kinetics , we used these conditions to determine the monomer flux rate from 25,000 MWCO membranes. Fig.…”

Section: Characterization Of Dialysis Membranesmentioning

confidence: 99%

Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics

Wilson

Adcock-Downey

Pusey

1996

Biophysical Journal

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Dialysis kinetics measurements have been made to study the effect of ionic strength on the dimerization of lysozyme in acidic solutions that lead to the growth of tetragonal lysozyme crystals. Using glutaraldehyde cross-linked dimers of lysozyme, we have determined that both monomers and dimers can escape from 25,000 molecular weight cutoff dialysis membranes with velocity constants of 5.1 x 10(-7) and 1.0 x 10(-7) s(-1) for the monomer and dimer species, respectively. The flux from 25K MWCO membranes has been measured for lysozyme in pH 4.0 buffered solutions of 1, 3, 4, 5, and 7% NaCl over a wide range of protein concentrations. Assuming that dimerization is the first step in crystallization, a simple monomer to dimer equilibrium was used to model the flux rates. Dimerization constants calculated at low protein concentrations were 265, 750, 1212, and 7879 M(-1) for 3, 4, 5, and 7% NaCl, respectively. These values indicate that dimerization increases with the ionic strength of the solution suggesting that aggregation is moderated by electrostatic interactions. At high protein concentrations and high supersaturation, the dimerization model does not describe the data well. However, the Li model that uses a pathway of monomer <-> dimer <-> tetramer <-> octamer <-> 16-mer fits the measured flux data remarkably well suggesting the presence of higher order aggregates in crystallizing solutions.

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“…The best technique discovered so far for the investigations of nucleation phenomena appears to be quasi-elastic light scattering (QELS). This has been applied to the investigation of crystallization for different macromolecular systems (for reviews, see Wilson, 1990;Kam, Shore & Feher, 1978;Carter, Baldwin & Frick, 1988;Bishop, Fredericks, Howard & Sawada, 1992;Kadima, McPherson, Dunn & Jurnak, 1991;Lorber, Skouri, Munch & Gieg6, 1993;Mikol, Hirsh & Gieg6, 1990). We recently applied QELS to the investigation of nucleation and crystallization phenomena in several large macromolecular assemblies that included satellite tobacco mosaic virus (STMV), apoferritin, ferritin and pumpkin seed globulin (Malkin, Cheung & McPherson, 1993;Malkin & McPherson, 1993a,b,c).…”

Section: Introductionmentioning

confidence: 99%

Light-scattering investigations of nucleation processes and kinetics of crystallization in macromolecular systems

Malkin¹,

McPherson²

1994

Acta Crystallogr D Biol Crystallogr

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Quasi-elastic light scattering (QELS) was used to investigate quantitatively the mechanisms of nucleation, postnucleation growth, and dissolution in ensembles of both crystalline and amorphous aggregates of satellite tobacco mosaic virus (STMV), ferritin, apoferritin and pumpkin seed globulin. At low supersaturation conditions, as described previously for small molecule crystallization, the metastable region was obtained. Under these conditions aggregation took place, but crystallization did not proceed and critical nuclei did not form over a long period of time. The critical solution supersaturation necessary to obtain crystals, o-= ln(c/s) where c and s are concentration and solubility of protein, varied from -0.1 for pumpkin seed globulin to -0.9 for STMV. For higher supersaturation conditions when aggregation processes leading to formation of crystals are not established immediately but after a certain induction period, the supersaturation-dependent critical nuclear size, R,, for different macromolecular systems was estimated from time-dependent sizedistribution analyses to be in the range of -10 3 for proteins such as pumpkin globulin to approximately 10 for virus particles. From the same data, the molar interracial free energy was deduced to be 3.3-9.2 kJ mol-t. These are believed to be among the first estimates for macromolecular crystals. Under conditions of moderate supersaturation where induction periods preceded the appearance of critical nuclei, the potential barriers for formation were estimated to be in the range 8.3-50 kJ mol-~. Growth and dissolution kinetics for pumpkin seed globulin were investigated. These experiments allowed determination of protein solubility versus solution temperature, protein and precipitant concentrations. Aggregation patterns which lead to crystal formation are distinctly different to those which produce an amorphous precipitate. The results provide additional evidence that QELS can be used to find general criteria that allow one to discriminate between conditions for a given protein system leading to crystalline or amorphous states at early stages of the aggregation process.

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Dynamic light scattering analysis of solutions from which lysozyme crystals grow

Cited by 20 publications

References 22 publications

Influence of polydispersity on protein crystallization: a quasi-elastic light-scattering study applied to α-amylase

Influence of polydispersity on protein crystallization: a quasi-elastic light-scattering study applied to α-amylase

Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics

Light-scattering investigations of nucleation processes and kinetics of crystallization in macromolecular systems

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