Crystallization and preliminary X-ray structural analysis used for characterisation of di-heme cytochrome c4

 

I. Tomčová  and   I. Kutá Smatanová

 

Institute of Physical Biology, University of South Bohemia, Nové Hrady, Czech Republic

Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Nové Hrady

 

Keywords:  crystallization of proteins; X-ray studies; metalloprotein structures studies

 

The cytochromes and the hydrogenases are ubiquitous proteins present in all living organisms and involved in a variety of intracellular processes that are essential for life. Most notable is their participation in electron transfer reactions, usually as components of a complex reaction pathway, necessary for the production of energy either through oxidation of metabolites or via photosynthesis [1]. The cytochromes consist of two heme molecules in single polypeptide chain with classical Cys–X–Y–Cys–His heme binding sites. It is the first cytochrome of its class that comes from an anaerobic organism. Due to their important function, it is of essential interest to study structural features of metalloenzymes using X-ray crystallography.

Cytochrome c4 (cyt c4) and hydrogenase from the purple sulphur photosynthetic bacterium Thiocapsa roseopersicina were isolated and purified according to [2]. Cyt c4 was crystallized using standard crystallization methods based on vapor diffusion [3] and advanced crystallization method based on the counter-diffusion [4]. Crystallization trials were performed at 20°C. The most suitable concentration of protein 10 mg/ml was found. The first suitable crystal growth was observed at pH 6.0 [Figure 1] using the addition of metal ions – Cu2+, Cd2+, Co2+, Ba2+ (Hampton Research Additive Screen).

 

 

 

 

 

 

 


Colored crossbred plates of holoprotein crystals with dimensions of approximately 200 x 50 x 30 μm grew within 3–4 days under several conditions.

The monocrystals of cyt c4 were tested at the home source diffractometer at LEC (University of Granada) and measured at synchrotron DESY (Hamburg), beamline X11 upto resolution 1.72 Å. Structure of cyt c4 will be solved using molecular replacement method.

This work is supported by grants of the Ministry of Education of the Czech Republic (by grants KONTAKT ME640, MSM6007665808, AVOZ60870520, MSM6007665808 and LC06010), by the EMBL-Hamburg Strategy Fund and by the ASCR (Institutional research concept AVOZ60870520).

 

[1]    T. Yamanaka: The Biochemistry Of Bacterial Cytochromes, Japan Scientific Societies Press, Tokyo (1992)

[2]    Cs. Bagyinka, R. M. M. Branca: unpublished data (2005)

[3]    T. M. Bergfors: Protein Crystallization. International University Line, La Jolla, USA (1999)

[4]    F. J. López-Jaramillo, J. M. García-Ruiz, J. A. Gavira, F. Otálora: J. Appl. Cryst. 34, 365-370 (2001)