Structural characterization of flavoprotein VrbA from Escherichia coli by using X-ray diffraction analysis

J. Wolfová^1,2, J. Carey³ and I. Kutá Smatanová^1,2

¹Institute of Physical Biology, University of South Bohemia České Budějovice, Zámek 136,
CZ-373 33 Nové Hrady, Czech Republic

²Institute of Systems Biology and Ecology, Academy of Science of the Czech Republic,
Zámek 136,CZ-373 33 Nové Hrady, Czech Republic

³Chemistry Department, Princeton University, Washington Rd and William St, Princeton,
NJ 08544-1009, USA

X-ray diffraction analysis is a main tool for structural analysis of biological macromolecules. Using of synchrotron radiation as a source is preferred for X-ray diffraction on protein crystals This study is concerned with the tryptophan (W)-repressor binding protein A (VrbA) from Escherichia coli, which belongs to a new family of multimeric flavodoxin-like proteins implicated in oxidative-stress defense. The suggested function is in agreement with the finding that the VrbA protein can be expressed also under the stress conditions [1]. The VrbA protein obtained its name with reference to its reported effect on the binding interaction of tryptophan repressor [2]. Nevertheless, it was shown later, that VrbA doesn’t influence the binding specifically [3], and thus its function in the living cells remains unclear. Based on the computational studies and biochemical experiments it was shown that the VrbA protein shares the open, twisted a/b fold with flavodoxins and its physiological cofactor is the flavin mononucleotide (FMN) as well [3, 4]. Unlike canonical flavodoxins the VrbA protein displays some interesting structural properties representing the new family, namely the structure of the flavin-binding pocket and multimerization. Structural characterization of the new protein family together with the understanding of the physiological role of VrbA protein in living organisms motivated our research of the VrbA protein using X-ray diffraction techniques. Moreover structural similarity with the proteins of the known functions (electron transfer in metabolic processes [8], protective effects on cells against free radicals [5-7], activation of cytotoxic drugs in cancer chemotherapy [9]) indicates the potential usage of the VrbA protein in pharmaceutics or medicine.

The pure VrbA apoprotein (protein without FMN) as well as the VrbA protein in complex with FMN were crystallized using standard vapor diffusion methods and counter-diffusion methods in single capillaries [10]. Optimization of crystallization conditions by using additives, especially Cd²⁺ and Li-citrate, led to getting single VrbA apoprotein crystals suitable for diffraction measurements [11]. The crystals diffracted to a resolution of 2.2Å at synchrotrons DESY (X13) in Hamburg (Germany), and Elletra (XRD1) in Trieste (Italy), but the diffraction data were not sufficiently good for solving structure. Additional optimization of crystal growth or preparation of the VrbA apoprotein crystals for diffraction analysis is necessary to obtain the data for solving the 3D-structure. The VrbA protein in complex with FMN showed better crystallizability then the protein without cofactor. This demonstrates the influence of cofactors on crystallization properties of proteins. Two crystallization conditions were found for this protein, both different from those used in the case of the VrbA apoprotein. The crystals of the liganded protein diffracted up to a resolution of 2.0Å at synchrotron DESY (X13) in Hamburg (Germany). The data sets collected at synchrotron were processed and the solving of the structure is in progress.

This work is supported by grant of the Ministry of Education of the CR (KONTAKT ME640) to I.K.S. and by NSF Grant INT-03-09049 to J.C. Grants MSM6007665808 and AVOZ60870520 are also acknowledged. Thanks to Jeroen Mesters, Jiří Brynda and Jan Dohnálek for their help with diffraction measurements of VrbA protein crystals and following data processing.

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