Heterooligomeric complexes of human small heat shock proteins

Abstract

Oligomeric association of human small heat shock proteins HspB1, HspB5, HspB6 and HspB8 was analyzed by means of size-exclusion chromatography, analytical ultracentrifugation and chemical cross-linking. Wild-type HspB1 and Cys mutants of HspB5, HspB6 and HspB8 containing a single Cys residue in position homologous to that of Cys137 of human HspB1 were able to generate heterodimers cross-linked by disulfide bond. Cross-linked heterodimers between HspB1/HspB5, HspB1/ HspB6 and HspB5/HspB6 were easily produced upon mixing, whereas formation of any heterodimers with participation of HspB8 was significantly less efficient. The size of heterooligomers formed by HspB1/HspB6 and HspB5/HspB6 was different from the size of the corresponding homooligomers. Disulfide cross-linked homodimers of small heat shock proteins were unable to participate in heterooligomer formation. Thus, monomers can be involved in subunit exchange leading to heterooligomer formation and restriction of flexibility induced by disulfide cross-linking prevents subunit exchange.

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

“…Combining NMR, ion-mobility native mass spectrometry, and EM, structural models of the most abundant oligomeric states (24-, 26-, and 28-mers) and a model for the conversion between them have been proposed, providing a deeper understanding of the polydispersity and dynamics of sHsp structures (Baldwin et al 2011b). Further increasing possible complexity, it has been found that various human sHsps may form mixed heterooligomers (Mymrikov et al 2012). The stable protein complexes formed between sHsp and substrate proteins also appear to be polydisperse (Stengel et al 2010), with complexes of pea Hsp18.1 and substrate protein luciferase comprised of variable numbers of both sHsp and substrate protein, covering over 300 different stoichiometries.…”

Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to their C-terminal ends

“…This complex was formed only if the mixture of two proteins was preincubated at an elevated temperature (Fig. 4a-c) (Mymrikov et al 2012;Datskevich et al 2012a). We measured the area of the peak of the wild-type HspB6 in the absence and in the presence of the wild-type HspB5 and, by this means, tried to estimate the approximate stoichiometry of the complex formed by these two proteins.…”

“…It is well known that the sHsp are able to form heterooligomers (Mymrikov et al 2012;Datskevich et al 2012a;Arrigo 2013). Therefore, it seems reasonable to analyze the effect of the yellow fluorescent protein attached to the C-terminal end of HspB6 on its ability to interact with the wild-type HspB1 and HspB5.…”

“…This makes it acceptable to use SEC to investigate the interaction of the Cterminal fluorescent chimera of HspB6 with two other sHsp. Mixed together and preincubated at an elevated temperature, the wild-type HspB1 and HspB6 are able to form two types of heterooligomeric complexes with M r 100 and 300 kDa (Bukach et al 2009;Mymrikov et al 2012;Datskevich et al 2012a) (Fig. 3a).…”

“…This domain is flanked by variable N-terminal and C-terminal regions that have important roles in the interaction of sHsp with protein targets, as well as in the formation of sHsp oligomers (Hilton et al 2012;Delbecq et al 2012). As a rule, sHsp form homooligomers or heterooligomers with a very flexible structure (Hilton et al 2012;Mymrikov et al 2012;Arrigo 2013). These oligomers can undergo rapid association and dissociation by exchanging their subunits and can change their intracellular location upon different stimuli (Bryantsev et al 2002;Clarke and Mearow 2013).…”