Mindy I. Davis Ph.D. National Institute of Health

Researchers apply acoustic liquid-transfer technology to protein crystallization.

Acoustic dispensing of compounds in dimethyl sulfoxide is becoming widely used for high-throughput assays. This technology uses sound waves to transfer a quantity of liquid from a donor plate up through the air to an inverted receiving plate. The liquid is retained in the wells, forming hanging drops. The receiving plate is normally then flipped back right-side up, and the assay then continues.

This article highlights a creative use of acoustic technology to initiate crystallization trials. The authors* point out that this technology could be used to set crystals in a 1536-well format in which the plate from which the precipitant is dispensed becomes the crystallization reservoir and the receiving plate, which is sealed to it, becomes the hanging drop surface and is left inverted. Alternatively, they also tested scenarios in which the reservoir of 1–2 μL is dispensed by the acoustic dispenser adjacent to the 5–10 nL protein droplet. The plate could then be sealed and left inverted with the drop and the reservoir existing side by side.

Acoustic dispensing for crystallization could allow for increased throughput and lower reagent and protein consumption by enabling rapid setup of very small volume crystallization trials, i.e., nanocrystallization. There are other techniques that have been used to set up small (<100 nL) crystallization trials, but they can suffer from artifacts, such as tip clogging or difficulty in harvesting the crystals.

The authors showed that the majority of the 284 commercial crystallization reagents tested, which can include quite viscous liquids, were transferred successfully by this method. Transfer was successful for 93% of the solutions tested, and the majority of the solutions that did not transfer effectively contained >50% 2-methyl-2,4-pentanediol. Overall the coefficient of variation (CV) for the volume transferred (5 nL) was quite good, with 73% of the reagents having a CV of <10%. The authors tried to add various oils onto the drops to minimize evaporation of the drops, but the transfer of the oils was problematic with the acoustic technology.

A diverse set of proteins were tested using the acoustic dispensing method, and the crystals that were obtained at day 15 (Figure) indicate that crystals can be successfully grown using this technique. The authors mention that for one previously uncrystallized protein complex, they were able to obtain crystals and solve the structure using this technique. They were able to obtain satisfactory crystals and solve the structure of their Fab-protein target using just 10 μL of total protein for a series of 20-nL test drops.

It will be interesting to see how easily diverse proteins will crystallize in this nanoliter environment. In particular, this technique could greatly increase the number of conditions that can be tested for proteins in which protein supply is limited. Crystallography can be quite useful for guiding medicinal chemistry optimization during drug discovery, and this technique might make crystal structures more accessible for projects in which protein limitations had previously precluded it.

Figure. Images collected on day 15 from crystallization drops of various protein samples. The total drop volume is double the protein value given. The volumes are representative and were not optimized as similar crystals appeared at the various volumes explored. (a) HCV helicase (50 nL). (b) Human serum albumin (50 nL). (c) HCV polymerase (30 nL). (d) HIV RT (15 nL). (e) ITK (30 nL). (f) Lysozyme (15 nL). HCV, hepatitis C virus; HIV, human immunodeficiency virus; RT, reverse transcriptase; ITK, interleukin 2–inducible T-cell kinase.

*Abstract from Acta Crystallographica Section D 2012, Vol. 68: 893–900

Focused acoustic energy allows accurate and precise liquid transfer on scales from picoliter to microliter volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nL. Only higher concentrations (>50%) of 2-methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanoliter crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format.

Mindy I. Davis, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Acta Crystallographica Section D titled “Nanolitre-scale crystallization using acoustic liquid-transfer technology.” Authors of the paper are Villaseñor AG, Wong A, Shao A, Garg A, Donohue TJ, Kuglstatter A, and Harris SF.

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