by Michael Collazo, Soheila Vaezeslami, and Sarah Burl
Pharmaceutical
Improving the Crystallization Process for Optimal Drug Development
acromolecular X-ray crystallography is an important and powerful tech- nique in drug discovery. Studying the specific interactions of a particular drug with its protein target at the atomic level can help improve the drug design process.
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Hundreds of different crystallization conditions need to be tested against a macromolecule of interest to grow crystals of sufficient size and quality and enable successful structure deter- mination. With advanced synchrotron X-ray sources, crystals as small as 10 µm or less can now be used for X-ray diffraction, but typically various conditions are tested to obtain crystals of 50 µm or larger in size, to make the data col- lection easier and more robust.
The crystallization process can often prove inefficient, with only one or a few crystals pro- duced from several hundred test conditions. Occasionally these crystals will be of sufficient quality for X-ray diffraction analysis and permit determination of the structure, which elimi- nates the need for further optimization of the conditions. However, in most cases the original “hits” may not form large enough crystals, only form precipitates, or generate poor or no diffraction at all. In these cases the crystal- lization conditions of the original hits need to be optimized. Obtaining these optimized and high-resolution diffracting crystals from targets of interest is vital to determining accurate mo- lecular structures of these molecules.
Crystallization process The optimal crystallization condition relies on
multiple and complex screening combinations. This can include the optimization of variables such as pH and concentrations of reagents and proteins in the hit condition, for example, by creating a gradient of the components across the crystallization plate.
A dedicated automated robot such as the dragonfly™ (Figure 1a) from TTP Labtech
(Melbourn, U.K.) can be used to set up these gradients. Optimization of 4–9 different com- ponents including diluent can be set up using the device’s 5- or 10-pipetting head format into a 48- or 96-well crystallography plate. Even with highly viscous fluids, the dragonfly is capable of accurately dispensing a minimum volume of 0.5 μL from a 10-mL reservoir tray with zero cross-contamination due to its positive dis- placement dispensing. The process is quick and easy, taking less than 5 min to make a gradient of four components in a 96-well plate.
However, if a dedicated optimization robot such as dragonfly is not available, the mosquito® Crystal (Figure 1b) (TTP Labtech), by itself, is capable of setting up gradients of components directly into the drops. A protein sample and each component of an original hit condition can be “multiaspirated” at different volumes in the same pipet tip, prior to being dispensed simultaneously, with additional mixing if required. The y-axis of the plate can be varied by loading eight different concentrations of a protein solution into a column of a protein reservoir strip, and concentrations of different components or pH can be varied in the x-axis.
This same concept can be applied for either the hanging- or sitting-drop method. Compared to optimization of hits manually, which are sub- ject to inherent manual pipetting errors, this method is much faster and more accurate, and will save on reagents.
Experiment 1: Optimizing
proteinase K crystals In an experimental study, developed by Michael Collazo, Lab Manager at the UCLA Macromolecular Crystallization Facility (Los Angeles, CA), proteinase K crystals were op- timized in a hanging-drop format using the mosquito Crystal. This was achieved by setting up a gradient in the drops and placing them over a reservoir of the original condition (0.1
AMERICAN LABORATORY • 11 • MAY 2014 a
b
Figure 1 – a) dragonfly and b) mosquito Crystal. M tris, pH 7.5, 1.2 M AmSO4 ) using the multi-
aspirate function. In this example, the protein concentration was varied in the y-direction (50 mg/mL to 30 mg/mL in 0.1 M tris HCl buffer, pH 7.5, in eight rows of a 96-well plate) and the con- centration of ammonium sulfate in each drop was varied from 0.3 M to 0.8 M across 12 col- umns in the x-direction. One-microliter drops (1:1 of protein to condition) were set up and left to equilibrate over the reservoir solution, the plates were imaged for crystal formation, and the conditions producing the best crystals were determined (Figure 2).
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