Multiwell Microbatch Crystallization on a Thermal Gradient

Abstract

A versatile and inexpensive microbatch crystallization apparatus with an integrated thermal gradient is introduced. The device is designed to fit onto automated microscope stages for timed image acquisition. The thermal gradient is established through an aluminum plate machined to house commercially available microplates with a standard 96-well footprint. The novelty of the design includes online monitoring of crystal growth without interfering with the established thermal gradient. The temperature stability and unidirectional temperature gradient were demonstrated. Hen egg white lysozyme (HEWL) was used as a model macromolecule to observe the effect of temperature variations on crystal habit and the number and size of crystals produced.

Controlling protein crystal growth rate by means of temperature

“…This equation constitutes the starting point in the formulation of our model accounting for the dependence on temperature of the growth rate of protein-type aggregations as, for instance, lysozyme aggregations in aqueous solutions [27,28,29]. This dependence has been reported in experiments that confirm the sensitivity of the growth of the aggregates to temperature conditions [1,2,3,4,8,9], and can be analyzed through an adequate choice of the Gibbs energy and the specific physical properties of each protein crystal/aggregate such as thermal expansion coefficient and surface energy.…”

“Hot” Macromolecular Crystals

“…The success in crystallization of biological macromolecules depends on many factors, and one of the most significant of them is temperature. , Changes in the temperature of crystallization can provide quick and reversible control of the supersaturation level because the solubility of protein is a function of temperature and may change dramatically even if the temperature change is small. , Protein solubility may either increase or decrease as temperature is increased, and this behavior may be significantly altered by both pH and the ionic strength of the solution . Not only can the temperature affect the thermodynamics of the protein solution in terms of the solubility and phase behavior of the solution, but it can also affect the kinetics by which crystals are nucleated and grown affecting crystal size, morphology, and quality.…”

“…Most researchers appear to restrict themselves to “room temperature” or to temperatures that can be easily obtained in a laboratory environment, such as 4 °C. However, some groups have developed systems to accurately regulate temperature for crystallization experiments. − Very little has been reported in the literature about crystals grown at elevated temperatures (e.g., above room temperature), though apoferritin was reported to produce significantly larger crystals in batch experiments as the temperature of crystallization was increased from 30 to 40 °C . In another example, “heat treatment” of a viral protein−ligand solution at 37 °C for 5−10 min, followed by incubation on ice and crystallization, yielded better-diffracting crystals of the complex …”

Dependence of Apoferritin Crystal Growth on Temperature and Cadmium Concentration

“…A multiwell microbatch crystallization device controlled the crystallization temperature and provided a thermal gradient with small temperature increments along the gradient. 15 The setup allowed testing of a 2-dimensional matrix of crystallization conditions involving temperature and cadmium concentration; apoferritin crystal growth was monitored inline at defined time intervals. Furthermore, the osmotic second virial coefficient was measured for certain protein solutions and compared to the crystallization outcomes.…”