The Preliminary X-ray Study Of Protein HMO2from Saccharomyces Cerevisiae

Background High-mobility group box (HMGB) proteins are a set ofnuclear nonhistone proteins that are found in a variety of eukaryotic species.The proteins, which have a highly conserved three-dimensional structure,play an important role in transcriptional regulation, DNA repair,recombination, differentiation and extracellular signaling. As a componentof the high-mobility group box proteins, HMO2is a24-kDa widespreadnuclear nonhistone protein. Furthermore, the high evolutionarilyconservation and widespread distribution of HMO2in eukaryotic speciesimply that HMO2has important biological functions, such asATP-dependent chromatin remodelling and DNA damage repair. However,as far as we know, the research about the structure of HMO2fromSaccharomyces cerevisiae has not been reported by now.Objectives: Firstly to resolve the HMO2’s three-dimensional structurefrom Saccharomyces cerevisiae, and then to research the HMO2’sbiochemical and physiological function with the structure information. Theresolved three-dimensional structure of HMO2can be applied to understandhow HMO2participate in both chromatin remodeling and DNA damagerepair.Methods: The HMO2gene was amplified from Saccharomycescerevisiae genomic DNA and inserted into pET22b to construct the plasmidpET22b-HMO2. And then the HMO2protein was purified by nickel ionaffinity chromatography column (Ni-NTA), anion exchangechromatography column (Resource Q) and gel exclusion chromatography (sieve chromatography Superdex200). The HMO2’s aggregation in thesolution was analyzed by gel exclusion chromatography. Both sitting-dropand hanging-drop vapour-diffusion methods are adopted to grow theHMO2crystal, and the crystal diffraction data were collected on theShanghai Synchrotron Radiation Facility (SSRF), and then itsthree-dimensional structure was resolved by the preliminary X-raycrystallographic studies.Results: The experiments which include constructing recombinantplasmid pET22b-HMO2, expressing and purifying the HMO2protein withthe purity of98%are sucessfully conducted. The size exclusionchromatography results showed HMO2protein exhibited as dimer in thesolution. Some good single crystals suitable to X-ray diffraction weresuccessfully obtained by the screening and optimization of protein crystals,and the X-ray diffraction results were collected on the BL17U1beamline atSSRF. The crystal of HMO2belonged to the space group P222or P212121,which has unit-cell parameters a=39.35, b=75.69and c=108.03. Basedon the space group and molecular weight of HMO2(about24kDa),solvent-content analysis showed that one molecule could be accommodatedper asymmetric unit, suggesting a VMvalue of3.193Da-1. However,multiple-wavelength anomalous dispersion method failed to resolve thethree-dimensional structure of HMO2protein.