Optimization of crystallization of biological macromolecules using dialysis combined with temperature control

Abstract

A rational way to find the appropriate conditions to grow crystal samples for bio-crystallography is to determine the crystallization phase diagram, which allows precise control of the parameters affecting the crystal growth process. First, the nucleation is induced at supersaturated conditions close to the solubility boundary between the nucleation and metastable regions. Then, crystal growth is further achieved in the metastable zone -which is the optimal location for slow and ordered crystal expansion -by modulation of specific physical parameters. Recently, a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature-precipitant-concentration phase diagrams has been constructed. Here, it is demonstrated that a thorough knowledge of the phase diagram is vital in any crystallization experiment. The relevance of the selection of the starting position and the kinetic pathway undertaken in controlling most of the final properties of the synthesized crystals is shown. The rational crystallization optimization strategies developed and presented here allow tailoring of crystal size and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination. research papers J. Appl. Cryst. (2020). 53, 686-698 Niels Junius et al. Optimization of crystallization of biological macromolecules 687 research papers J. Appl. Cryst. (2020). 53, 686-698 Niels Junius et al. Optimization of crystallization of biological macromolecules 689

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

“…For most proteins, the precise quantitative phase diagram (or just a qualitative diagram) has not yet been obtained due to the lack of experimental devices capable of accurately measuring protein concentration (or just of observing/ detecting the crystallization process in real time) during crystallization experiments 18 . As a result, it is often not possible to design the experiment in such a way that crystallization begins in the optimal area of the phase diagram, in the vicinity of the metastable zone.…”

“…buffer. In such a case, it is crucial to set the pH according to the temperature at which the experiment is performed 18 . It should also be noted that protein dissolution takes significantly less time (from a few minutes to a few hours) compared to protein crystal growth (from a few hours to a few days).…”

“…The optimization of crystal growth with OptiCrys is based on temperature-precipitant concentration phase diagrams. In the case of proteins with solubility as a direct function of temperature, it is possible to make use of the salting-out regime 18 . This is where increasing the ionic strength of the solution, which can be visualized using protein-precipitant phase diagrams, decreases the solubility of the protein.…”

“…Likewise, proteins with inverse solubility can make use of the salting-in regime 18 . Nucleation occurs in the nucleation zone, in the vicinity of the metastable zone, and crystal growth then takes place in the metastable zone of the phase diagram until the protein concentration reaches the solubility limit.…”

Mixing Salts and Poly(ethylene glycol) into Protein Solutions: The Effects of Diffusion across Semipermeable Membranes and of Convection

“…[11][12][13][14][15] Dialysis is the basis of the crystallization apparatus developed recently to monitor and control the crystallization processes in situ. 16,17 We present both experimental data, and quantitative models, and show how to estimate rates of the mass transport processes, and hence which processes are rate limiting. The processes are diffusion both in solution and across a membrane, as well as convection in solution.…”