Investigation of Biochemical Structure and Functions of the E. coli Protein WrbA

I. Kishko ^{1, 2}, J. Carey ³, R. Ettrich ^{1, 2}

¹Institute of Physical Biology, USB CB, 37333 Nove Hrady, Czech Republic

²Institute of Systems Biology and Ecology, AS CR, 37333 Nove Hrady, Czech Republic

³Department of Chemistry, Princeton University, Princeton, New Jersey 08544-1009, USA

Kishko@greentech.cz

The structure of the E.coli flavoprotein WrbA previously showed it is structurally related to eukaryotic NADH:quinone oxidoreductases (Nqos)[1]. Those enzymes have many unusual kinetic properties and their physiological function is not clear [2, 3].

WrbA and Nqos can transfer two electrons at a time from NADH to quinone acceptors. The electron transfer kinetics can be observed spectrophotometrically under steady-state conditions [1, 4, 5]. This assay is used to measure the rates of WrbA electron transfer and to evaluate different compounds that might function as the true physiological electron donors or acceptors. For the co-crystallization experiments we used standard equipment for crystallization, and varied the conditions that were established in our group for the holo protein crystals.

The results of this work demonstrate unusual two-plateau behaviour on the substrate concentration-dependence plots for NADH or benzoquinone. The experiments show that WrbA activity increases upon addition of membrane-mimicking detergents, and they demonstrate the ability of the protein to inactivate reversibly by shifting temperature from 5 to 25 ^oC. These properties are similar for the Nqos but have not been explained. Microcrystals of WrbA protein were crystallized using the sitting-drop vapour-diffusion technique [6]. Future studies with WrbA have the aim to explain kinetic properties in molecular terms and to create crystal good quality for diffraction analysis.

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5. Noll G, Kozma E, Grandori R, Carey J, Schodl T, Hauska G, Daub J. Langmuir, 22, (2006), 2378-83.

6. A. Ducruix & R. Giegé, Crystallization of Nucleic Acids and Proteins: A Practical Approach, 2nd ed. Oxford: Oxford University Press, (1999).

This project was supported by grants: LC06010 (Ministry of Education of the Czech Republic) and GAJU 079/2008/P.