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: http://www.biochem.bio.msu.ru/publications/publication.php?pubmedID=22531625
Дата изменения: Unknown Дата индексирования: Thu Feb 27 21:55:43 2014 Кодировка: |
| Title: | Utilization of fluorescent chimeras for investigation of heterooligomeric complexes formed by human small heat shock proteins. |
| Authors: | Datskevich PN; Mymrikov EV; Gusev NB |
| Publication: | Biochimie. 2012 Aug;94(8):1794-804. doi: 10.1016/j.biochi.2012.04.012. Epub 2012 Apr 17. |
| PubmedID | 22531625 |
| Abstract | |
| Fluorescent chimeras composed of enhanced cyan (or enhanced yellow) fluorescent proteins (ECFP or EYFP) and one of the four human small heat shock proteins (HspB1, HspB5, HspB6 or HspB8) were expressed in E. coli and purified. Fluorescent chimeras were used for investigation of heterooligomeric complexes formed by different small heat shock proteins (sHsp) and for analysis of their subunit exchange. EYFP-HspB1 and ECFP-HspB6 form heterooligomeric complex with apparent molecular weight of approximately 280 kDa containing equimolar quantities of both sHsp. EYFP-HspB5 and ECFP-HspB6 formed heterogeneous oligomeric complexes. Fluorescent proteins inside heterooligomeric complexes formed by HspB1/HspB6 and HspB5/HspB6 chimeras are closely located, making possible effective fluorescence resonance energy transfer (FRET). Neither the wild type HspB8 nor its fluorescent chimeras were able to form stable heterooligomeric complexes with the wild type HspB1 and HspB5. Homo- and hetero-FRET was used for analysis of subunit exchange of small heat shock proteins. The apparent rate constant of subunit exchange was temperature-dependent and was higher for HspB6 forming small oligomers than for HspB1 forming large oligomers. Replacement induced by homologous subunits was more rapid than the replacement induced by heterologous subunits of small heat shock proteins. Fusion of fluorescent proteins might affect oligomeric structure of small heat shock proteins, however fluorescent chimeras can be useful for investigation of heterooligomeric complexes formed by sHsp and for analysis of kinetics of their subunit exchange. | |