ИСТИНА

Small heat shock proteins (sHsp) HspB1, HspB5, HspB6 and HspB8 are ubiquitously expressed in human tissues, where they function as molecular chaperones preventing aggregation of misfolded or partially denatured proteins. sHsp also participate in regulation of different vital processes such as protein folding and degradation, cell proliferation, apoptosis, cytoskeleton regulation. Fluorescent chimeras are often used for investigation of location and functioning of different protein in vivo, however up to now biochemical properties of sHsp fluorescent chimeras were not thoroughly analyzed. We investigated properties of chimeras consisting of one of the four human sHsp and the enhanced cyan or yellow fluorescent protein (FP) fused to the N- or C-ends of sHsp. The wild type HspB1 and HspB5 form large oligomers containing more than 20 subunits. Fluorescent chimeras of these proteins form homooligomers containing smaller number of subunits and number of subunits and stability of oligomers decreased in the order: HspB1 WT> HspB1-FP> FP-HspB1, HspB5 WT> HspB5-FP> FP-HspB5. Chimeras of HspB6 and HspB8 form dimers or small oligomers, as well as the wild type proteins. The chaperone-like activity of fusion proteins was different from that of wild type proteins. Homo- and hetero-FRET was used for analyzing subunit exchange of different sHsp and rate of exchange was estimated. It is concluded that the fusion of fluorescent protein alters the structure and properties of sHsp and that this effect depend on location of fluorescent protein inside of the chimeras. This investigation was supported by Russian Foundation for Basic Science.