title: Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to their C-terminal ends.
abstract: Recombinant chimeras of small heat shock proteins (sHsp) HspB1, HspB5, and HspB6 containing enhanced yellow fluorescent protein (EYFP) attached to their C-terminal ends were constructed and purified. Some properties of these chimeras were compared with the corresponding properties of the same chimeras containing EYFP attached to the N-terminal end of sHsp. The C-terminal fluorescent chimeras of HspB1 and HspB5 tend to aggregate and form a heterogeneous mixture of oligomers. The apparent molecular weight of the largest C-terminal chimeric oligomers was higher than that of the corresponding N-terminal chimeras or of the wild-type proteins; however, both homooligomers of N-terminal chimeras and homooligomers of C-terminal chimeras contained fewer subunits than the wild-type HspB1 or HspB5. Both N-terminal and C-terminal chimeras of HspB6 form small oligomers with an apparent molecular weight of 73-84 kDa. The C-terminal chimeras exchange their subunits with homologous wild-type proteins. Heterooligomers formed by the wild-type HspB1 (or HspB5) and the C-terminal chimeras of HspB6 differ in size and composition from heterooligomers formed by the corresponding wild-type proteins. As a rule, the N-terminal chimeras possess similar or slightly higher chaperone-like activity than the corresponding wild-type proteins, whereas the C-terminal chimeras always have a lower chaperone-like activity than the wild-type proteins. It is concluded that attachment of EYFP to either N-terminal or C-terminal ends of sHsp affects their oligomeric structure, their ability to form heterooligomers, and their chaperone-like activity. Therefore, the data obtained with fluorescent chimeras of sHsp expressed in the cell should be interpreted with caution.
authors.affiliation: Department of Biochemistry, School of Biology, Moscow State University, Moscow, 119991, Russian Federation.
authors.forename: Petr N
authors.initials: PN
authors.lastname: Datskevich
authors.forename: Nikolai B
authors.initials: NB
authors.lastname: Gusev
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D050888:Heat-Shock Proteins, Small / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D050888:Heat-Shock Proteins, Small; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria