Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to...

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.