At the beginning of March, the University of Cape Town organized a workshop called CCP4 Crystallographic School in South Africa. Due to the current situation, it was of course in the online environment and Ing. Andrea Schenkmayerová Ph.D. from Loschmidt laboratories of Faculty of Science MU and the International Clinical Research Center of St. Anne’s University Hospital Brno (FNUSA-ICRC) achieved great success. The researcher of the Protein Engineering reserach team won the award for the best poster entitled Structural analysis of a haloalkane dehalogenase from subfamily HLD-III.
In general, crystallography is a scientific discipline that deals primarily with the study of the arrangement and bonding of atoms in crystals and the study of the geometric structure of crystal lattices. Although most of us imagine a crystal such as a grain of salt, the modern concept of a crystal is based directly on the characteristics of the internal structure at the level of atoms and not on its external shape. The crystalline state of matter is more energetically advantageous and at present we can get not only minerals but also metal alloys or organic molecules into this state. The importance of crystallography is also underlined by the fact that 32 Nobel Prizes have so far been awarded for research and related results.
Macromolecular crystallography deals with the study of the structure and spatial arrangement of biological macromolecules (eg proteins, DNA) and their complexes, which is key to understanding their function in organisms. A detailed understanding of the structure and function of biological macromolecules is then key to understanding complex cellular processes, their homeostasis but also their pathological manifestations.
Crystallography is also used to research new proteins and their inhibitors, which could be used, for example, as drugs. The therapeutic effect is influenced, inter alia, by the shape of the molecules of the therapeutic component, so that crystallography functions here as a tool for obtaining information about the shape of the molecules. However, we would not find a classic optical microscope in this area, the light has too long wavelength – for microscopy at the molecular level devices using for example, X-rays. In general, the best drug is one whose molecule fits into a suitable binding site in the macromolecule and thus affects its biological activity.
Ing. Andrea Schenkmayerová Ph.D. in her work focused on hitherto structurally unexplored enzymes from the haloalkane dehalogenase family. These enzymes have an interesting property – they catalyze the cleavage of carbon-halogen bonds to form the corresponding alcohol, halogen anion and proton. Due to these properties, these enzymes are used in various biotechnological applications. In addition to haloalkane dehalogenase activity, lactone decarboxylase activity has been found in some of these enzymes in recent years, prompting a broad scientific discussion of the natural biological function of these enzymes and how they evolved during evolution.
“It was started by my colleague Ing. Klaudia Chmelová and after she left for maternity, I started doing it, “said Schenkmayerová. “It was a real challenge, because so far no one in the world has been able to determine the structure of the enzyme from the HLD-III subgroup, because it forms heterogeneous oligomeric structures, which makes their structural analysis very impossible. In our laboratory, systematic work has succeeded in developing a method by which we are able to prepare relatively homogeneous enzyme preparations, which opened the way for their structural analysis using cryo-electron microscopy and X-ray crystallography. Although we eventually managed to prepare crystals of this enzyme and collect quality crystallographic data, we still had trouble solving the structure due to the atypical internal arrangement of the crystal and the lower resolution of the obtained crystallographic data.”
She signed up for the workshop with work that still needed to be completed, and with the help of lecturers, she finally succeeded. It was a truly international collaboration, on the result contributed for example, professor Kay Diederichs from the University of Konstanz or professor Randy J. Read and Dr. Tristan Croll from the University of Cambridge. Important data were obtained in CEITEC laboratories and measurements were also performed on a Swiss Light Source synchotron device in Switzerland.
“This is a perfect example of an integrated approach in structural biology, which combines several experimental approaches at the same time so that it is possible to solve the structure of biomolecules when one technique is not enough. The original, say, wild-type protein formed various types of oligomers and we failed to crystallize for a long time. Using protein engineering methods, we prepared a stabilized form of the enzyme that did not produce so many different types of oligomers, and we were able to crystallize it. Despite all the difficulties, the workshop managed to solve the crystal structure of this unexplored enzyme, which will help us understand the biological function of these very interesting biocatalysts, “described Schenkmayerová.
A manuscript of the publication is currently being prepared, we will inform you as soon as it is ready.
Fig. 1: Ing. Andrea Schenkmayerová PhD. with her leader RNDr. Ing. Martin Marek Ph.D.
Fig. 2: Photo of protein crystal