NMR study of haloalkane dehalogenase DhaA: resonance assignment and relaxation

V. Zapletal¹, M. Zachrdla¹, P. Srb¹, L. Žídek¹, R. Chaloupková²

¹NCBR, Faculty of Science and CEITEC, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

²Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno,Czech Republic

vojtis@mail.muni.cz

Nuclear magnetic resonance (NMR) is a method well suited for studies of biomacromolecules at the atomic level. Wild type haloalkane dehalogenase DhaA, obtained from the bacterium Rhodococcus rhodochrous, is a 33.5 kDa enzyme capable of hydrolytic biodegradation of aliphatic alogen-hydrocarbons yielding a halide, a proton and a primary alcohol. The goal of the project was to describe the molecular motions of DhaA. As the first step, it is necessary to assign the resonance frequencies. The sample for assignment was uniformly triply labeled with ²H, ¹³C and ¹⁵N isotopes and for relaxation study was doubly labeled with ²H and ¹⁵N isotopes. The experiments utilizing transverse relaxation-optimized spectroscopy (TROSY) were performed. The assignment of 222 residues was achieved, which represents approximately 90 % of the observed backbone amide peaks in the ¹H-¹⁵N TROSY spectrum. TROSY-based R₁, R₂ and ¹⁵N-{¹H} steady-state NOE experiments were carried on the 950 MHz US² Bruker spectrometer and they were used to provide information on molecular motions on the fast time scale.