Influence of additives on crystallization of the flavoprotein WrbA

J. Wolfová1, J. Carey2 and  I. Kutá Smatanová3

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<aff><oid id="1">Institute of Physical Biology, University of South Bohemia at Ceske Budejovice, Zamek 136,  373 33 Nove Hrady, <cny>Czech Republic

 </cny></aff><aff><oid id=" 2<aff><oid id="">Chemistry Department, Priceton University, Washington Rd and William St, Princeton,  NJ 08544-1009, <cny>USA</cny></aff></aug> 

 3</cny></aff>">Institute of Physical Biology, University of South Bohemia at Ceske Budejovice and Institute of Landscape Ecology, Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove  Hrady, <cny>Czech Republic

 

A novel flavodoxin-like protein, tryptophan (W)-repressor binding protein A, WrbA, was first identified as an Escherichia coli stationary-phase protein enhancing the binding interaction between DNA and the tryptophan repressor (TrpR), which regulates the biosynthesis of tryptophan in procaryotes. Later work [1] showed that WrbA doesn't specifically influence the binding of TrpR to DNA and thus left the physiological role of WrbA unclear. According to sequence analysis and homology modelling, WrbA was identified as the founding member of a new protein family, sharing the open, twisted a/b fold typical for flavodoxins [2]. The biochemical and biophysical studies of purified WrbA protein [1] revealed that the WrbA protein binds flavin mononucleotide (FMN) similarly to flavodoxins, but the affinity of WrbA for FMN is much lower. This may be associated with the structural differences in the flavin-binding pocket predicted by the computer analysis. Analytical ultracentrifugation in combination with the size-exclusion chromatography showed the multimeric character of WrbA protein in solution. WrbA is apparently the first characterized case in which multimerization is associated directly with the flavodoxin-like domain itself. In all other multimeric flavodoxins the flavodoxin-like domain is fused to a multimerization domain [3]. WrbA protein and its homologs thus present a unique family among the typical flavodoxin-like proteins. Structural analysis may aid in understanding these unique properties and may reveal the physiological role of WrbA in the living organisms. This was a motivation for searching of diffraction-quality crystals.

Crystallization conditions for growing of WrbA apoprotein crystals were found using standard and advanced crystallization techniques [4]. As the WrbA crystals grew as twinned plates additives were used to improve the quality of crystals. Crystals suitable for X-ray diffraction measurements grown in capillaries as well as in sitting drops will be measured at synchrotron at cryotemperature and the diffraction data will be used for solving protein structure in the future.

Limited proteolysis [5] of WrbA apoprotein led to preliminary identification of folded substructures and flexible parts of protein structure. Further analysis of the fragments gained by the proteolytic digestion will serve as a competent accessory  to X-ray structure.

 

This work is supported by grant of the Ministry of Education of the Czech Republic (project KONTAKT ME640 to I.K.S.), by grants MSM6007665808 and AVOZ60870520, and by NSF grant INT-03-09049 to J.C.

 

1. R. Grandori, P. Khalifah, J.A. Boice, R. Fairman, K. Giovanielli & J. Carey,  J. Biol. Chem., 273 (1998) 20960-20966.

2. R. Grandori & J. Carey, Trends Biochem. Sci., 19 (1994) 72.

3. J. Ostrowski, M.J. Barber, D.C. Rueger, B.E. Miller, L.M. Siegel & N.M. Kredich, J. Biol. Chem., 264 (1989) 15796-15808.

4. J. Lopez-Jaramillo, J.M. Garcia-Ruiz, J.A. Gavira & F. Otalora, J. Appl. Cryst., 34 (2001) 365-370.

5. J. Carey, Methods Enzymol., 328 (2000) 499-514.