Influence of additives on crystallization of the flavoprotein WrbA
</snm></au><orf id="<au><fnm>
<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.