Article pubs.acs.org/crystal
Metal-Assisted and Microwave-Accelerated Evaporative Crystallization: Proof-of-Principle Application to Proteins Kevin Mauge-Lewis,† Adeolu Mojibola,† Eric A. Toth,‡ Muzaffer Mohammed,† Dereje Seifu,§ and Kadir Aslan*,† †
Department of Chemistry and §Department of Physics, Morgan State University, 1700 East Cold Spring Lane, Baltimore, Maryland 21251, United States ‡ Department of Biochemistry and Molecular Biology, Marlene and Stewart Greenebaum Cancer Center, Institute for Bioscience and Biotechnology Research, and Center for Biomolecular Therapeutics, University of Maryland at Baltimore, 9600 Gudelsky Drive, Rockville, Maryland 20850, United States S Supporting Information *
ABSTRACT: Rapid crystallization of a model protein, i.e., lysozyme, on blank and silvered circular crystallization platforms with 21 sample capacity, using the metal-assisted and microwave-accelerated evaporative crystallization (MA-MAEC) technique is described. The effectiveness of the MA-MAEC technique for the crystallization of lysozyme was compared to the conventional crystallization technique (i.e., at room temperature without microwave heating) based on the following parameters: crystallization time, crystal size, crystal number, and crystal quality. Using silvered platforms, the growth of lysozyme crystals was concluded within 857 ± 31 min and 565 ± 64 min, at room temperature and using the MA-MAEC technique (microwave power level 1 or duty cycle of 3 s in a kitchen microwave oven), respectively. On blank platforms (silver is omitted), the growth of lysozyme crystals was concluded within 1190 ± 14 min and 955 ± 50 min, at room temperature and using microwave heating. The largest sizes of lysozyme crystals (200 ± 50 μm) were grown on silvered platforms and microwave heating at power level 1. In addition, superior intraplatform and interplatform repeatability of growth of lysozyme crystals was observed with silvered platforms and microwave heating at power level 1. Although the same well-defined tetragonal shapes of lysozyme crystals were observed at both room temperature and using the MA-MAEC technique, the quality of lysozyme crystals was found to slightly deteriorate with the use of microwave heating.
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the lipid film and incubated for several hours. The crystal quality was shown to improve as lipid and detergent concentration was increased, and crystals grew to their full size within 2−4 weeks.3 Existing high-throughput crystallization technologies have improved prospects somewhat, but there is still considerable room for improvement. Any technology that produces diffraction-quality crystals with a higher success rate in a shorter time frame will move the field of structural biology forward significantly. In particular, increasing the rate of crystal production will allow investigators to take full advantage of existing high-throughput diffraction screening and data collection platforms. Structure-based drug design, in which a given target might need to be cocrystallized with hundreds of lead compounds, could derive a tremendous benefit from a rapid crystallization protocol with high success rates. The Aslan Research Group has recently demonstrated a crystallization technique, called metal-assisted and microwaveaccelerated evaporative crystallization (MA-MAEC),11 which is based on the combined use of plasmonic nanostructures and
INTRODUCTION Biological macromolecules are complex polymers, which makes the process of forming peptide and protein crystals extremely complex. Moreover, peptide and protein crystals typically contain at least 50% solvent molecules, making the contacts that form the crystal lattice weak relative to those of inorganic crystals. Therefore, crystallization experiments are largely empirical and unsuccessful. The crystallization of important peptides and proteins therefore remains a major roadblock to structural studies and structure-based drug design.1−6 In response to the need for efficient crystallization techniques, several techniques have been developed, such as microbatch crystallization,7,8 hanging drop vapor diffusion crystallization,9,10 and nanodroplet crystallization,6 just to name a few. In nanodroplet crystallization, a robotic workstation is used for the optimization of the protein crystallization process at small volumes, between 20 and 100 nL, where the time required for a 50 μm-sized crystal to form was 18.0 MΩ·cm resistivity at 25 °C) obtained from Millipore Direct Q3 system, except when stated otherwise. Methods. (1). Preparation of Lysozyme Solution. We employed a procedure recommended by the vendor: lysozyme powder (60 mg) was dissolved in 1 mL of sodium acetate (pH 4.6) and was mixed with 500 μL of crystallization reagent in a clean glass vial. The crystallization solution was later extracted using a sterile syringe (0.8 mm × 40 mm) and then slowly filtered back into the vial through a sterile syringe filter (ValuPrep, 25 mm cellulose acetate, 0.2 μm). 3213
DOI: 10.1021/acs.cgd.5b00334 Cryst. Growth Des. 2015, 15, 3212−3219
Crystal Growth & Design
Article
Figure 1. Real-color photograph of a (A) blank circular crystallization platform with and without a polymer cover (
