Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Madison Piassek - Georgian Court University, Lakewood, New Jersey, USA
Co-Author(s): Iuliia Iermak and Ivana Kuta-Smatanova, Faculty of Science, University of South Bohemia in Ceske Budejovice, Ceske Budejovice, Czech Republic
Proteins are a large class of macromolecules that participate in various cellular processes. Knowledge of three-dimensional protein structures is essential for full understanding of their functions. The principal technique for the structural characterization of proteins is X-ray diffraction analysis of protein crystals. The bottleneck of this technique is obtaining crystals of sufficient diffraction quality. Proteins are crystallized from mixtures that contain various precipitants (such as inorganic salts, organic polymers, etc.) creating conditions for spontaneous nucleation and crystal growth. Optimization of protein crystals aims to improve size and diffraction quality of crystals, and may involve variation of protein and precipitant concentrations, pH, temperature, or use of additives. Additives include a wide range of small molecules intended to enhance intermolecular contacts between protein molecules. Recently, ionic liquids (ILs) have attracted attention as potential additives for protein crystallization. ILs are organic salts which are liquid at room temperature. They exhibit a variety of properties that make them attractive solvents for biomaterials, such as the ability to participate in ionic, hydrophobic, and hydrogen bond interactions. ILs demonstrate localized structuring about each ion compared to materials of disassociated ions, setting them apart from salt solutions. Until recently, there has been little effort to utilize ILs for protein crystallizations. Several studies have been carried out using ILs in protein crystallization, but with no conclusions that could guide one in their use. Presented work aimed to investigate the impact of ILs as protein crystallization additives in order to advance the crystallization process. A panel of 24 commercially available ILs were tested as additives for several enzymes previously structurally characterized by our group, including the wild type and mutant glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH). Sitting-drop vapor diffusion crystallization experiments revealed that addition of ILs tended to result in precipitation of proteins. Nevertheless, for one of the conditions tested, crystals of the wild type TaAlDH were obtained. X-ray diffraction analysis showed alteration of the space group from P21 to C222 while resolution was not drastically changed. Results suggest subtle changes in solution conditions (such as pH, ionic strength, etc.) likely provided a change in crystal growth kinetics. However, it would be preliminary at this point to extrapolate specific effects of ILs to a broader range of proteins. Further work on evaluating the effects of ILs on protein crystallization, including analysis of the structures of proteins crystallized in the presence of ILs, is in progress.
Funder Acknowledgement(s): This work was carried out during summer 2016 at the Center for Nanobiology and Structural Biology in Nove Hrady, Czech Republic, supported by NSF award DBI13-58737 to J. Carey. I would like to express my thanks to Iuliia Iermak for valuable advice in the study and kind help with diffraction data collection. Support from Ivana Kuta-Smatanova and the University of South Bohemia in Ceske Budejovice is also appreciated.
Faculty Advisor: Jannette Carey, email@example.com
Role: I carried out all crystallization experiments involving ionic liquids and the wild type and mutant glyceraldehyde dehydrogenase from Thermoplasma acidophilum. I obtained crystals of the wild type in one of the ionic liquids tested, and these crystals were of sufficient diffraction quality.