Combination of X-ray diffraction analysis
and limited proteolysis used in structural study of flavoprotein WrbA
J. Wolfová1,2, J. Carey3 and I. Kutá Smatanová1,2
of Physical Biology, University of South Bohemia České Budějovice, Zámek 136,
CZ-373 33 Nové Hrady, Czech Republic
of Systems Biology and Ecology, Academy of Science of the Czech Republic,
Zámek 136,CZ-373 33 Nové Hrady, Czech Republic
Department, Princeton University, Washington Rd and William St, Princeton,
NJ 08544-1009, USA
The tryptophan (W)-repressor binding protein A (WrbA) identified as an Escherichia coli stationary-phase protein belongs to a new family of multimeric flavodoxin-like proteins implicated in oxidative-stress defense. Based on the computational studies and biochemical experiments it was shown that the WrbA protein shares the open, twisted a/b fold with flavodoxins and its physiological cofactor is the flavin mononucleotide (FMN) as well [1, 2]. Since WrbA is largely uncharacterized with respect to both molecular and physiological functions and displays some interesting structural properties representing the new family, the present effort is aimed at structural characterization by X-ray diffraction analysis and limited proteolysis.
The WrbA apoprotein was successfully crystallized using standard vapor diffusion methods and counter-diffusion methods in single capillaries . Optimization of crystallization conditions by using additives, especially Cd2+ and Li-citrate, led to getting single crystals suitable for diffraction measurements . 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 crystallization conditions of the WrbA apoprotein is in progress. The WrbA protein in complex with the cofactor, FMN, has also been crystallized, but the crystallization has to be optimized yet.
For a complementary investigation of the folding architecture of the WrbA protein the proteolytic dissection method, called limited proteolysis, was applied. It is an important method used for identification and characterization of the folded substructures of proteins and their stability . Although the WrbA protein contains many potential cleavage sites for the proteases used, the intact protein was attacked by proteases only at several specific sites, which resulted in several protein fragments identified by the SDS-PAGE. The presence of the folded structure in these fragments was confirmed by CD spectroscopy. This result is in agreement with the theoretical assumption that only the accessible cleavage sites lying in the incompletely ordered parts of protein structure are attacked by proteases while the cleavage sites buried inside the folded structure remain intact. By the limited proteolysis a fragment of nearly the same size as the intact protein was generated. This result indicates that the WrbA protein contains a flexible part at one end. Removing of this part of protein might improve crystallizability of the protein. The experiments showed also that the FMN binding increases the protein stability, which was indicated by the longer resistance of the intact protein to protease activity. Sequencing of the fragments produced by proteolytic digestion is under way, promising to shed light on the folding of WrbA protein and to identify the part of protein causing the crystallization difficulties. The results reported here show that the limited proteolysis could serve as a competent accessory to X-ray diffaction study.
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 and Maurizio Polentarutti for their help with diffraction measurements of WrbA apoprotein crystals.
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