Crystallization of novel haloalkane dehalogenase from Glaciecola Agarilytica NO2 and atomic force microscopy applications in macromolecular crystallization

D. Malakhova1, 2, I. Iermak1, R. Chaloupkova3, I. Kuta Smatanova1, 4 and D. Stys2

1University of South Bohemia, Faculty of Science, Branišovska 31, 370 05, Ceske Budejovice, Czech Republic

2University of South Bohemia, Faculty of Fisheries and Protection of Waters, Institute of Complex Systems, Zamek 136, 373 33 Nove Hrady, Czech Republic

3Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, 625 00 Brno, Czech Republic

4Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady, Czech Republic

dmalakhova@frov.jcu.cz

The main bottleneck of solving protein structures by means of X-ray crystallography is obtaining good quality protein crystals. Atomic force microscopy (AFM) can provide information about growth and perfection of crystals, impurity effects, and defect formation, furthermore, AFM images can give low - resolution phase information which could enhance X-ray diffraction analysis (Malkin et al., 2004). This type of probe microscopy allows topography of soft biological samples to be imaged in situ in physiological condition with molecular resolution. Therefore combination of AFM and X-ray diffraction techniques will be used as a toolkit for macromolecular crystallography.

Microbial enzymes such as haloalkane dehalogenases have attracted significant interest because of their ability to catalyze the irreversible hydrolysis of wide range of halogenated compounds to the corresponding alcohol, halide ion and proton. These enzymes play an important role in biodegradation of halogenated compounds, which appear to be environmental pollutants (Newman et al., 1999, Janssen, 2004). Novel haloalkane dehalogenase DgaA was isolated from psychrophilic and moderate halophilic organism Glaciecola agarilytica NO2 found in marine sediment collected from the East Sea, Korea (Yong et al., 2007).

Microcrystals of DgaA grew in several conditions of JCSG-plus and Structure Screen 1&2 (Molecular dimensions Ltd, UK) after initial screening. Optimization including variation of salt, protein and polymer concentrations resulted in certain improvement of crystals. The best needle-shaped crystals with size about 0.8 x 0.039 mm and plate clusters with plate size of approximately 0.03 x 0.2 mm were obtained. Further crystals preparation for AFM experiments is in progress.

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This research was supported by the GAJU 134/2013/Z,), by the GACR 207/12/0775 and by the Ministry of Education, Youth and Sports of the Czech Republic - projects CENAKVA (No. CZ.1.05/2.1.00/01.0024) and CENAKVA II (No. LO1205 under the NPU I program).