In particular, no information is available on the multimeric structure and the

smHsp amino acids involved in this membrane-stabilizing effect. We exploited the fact that Lo18 WT and its respective proteins with amino acid substitutions can be overexpressed in E. coli, to analyse their abilities BYL719 in vitro to modulate membrane fluidity in E. coli whole cells at 50 °C. We investigated the association between Lo18, including its three proteins with amino acid substitutions, and the membrane fraction of E. coli by Western blotting experiments. All the proteins studied were well associated with the E. coli membrane fraction (data not shown). Moreover, the malate dehydrogenase activity was controlled to validate the separation of both fractions. The changes in membrane

fluidity expressed in anisotropy percentages were compared with the initial anisotropy value (Fig. 4). The results indicated that the increase in temperature was followed by Vemurafenib cell line an increase in membrane fluidity. These first variations were observed equally for all strains (up to 5 min), but the recovery of the initial level of fluidity varied, depending on the strains. The strains producing Lo18 WT, V113A and A123S regulated membrane fluidity to reach, respectively, 100%, 91% and 90% of the initial value 30 min after the heat shock (Fig. 4). By contrast, the strain overexpressing Y107A was unable to regulate membrane fluidity and displayed characteristics similar to the control (with a fluidity recovery of 67% and Gefitinib datasheet 75%, respectively). Thus, the Y107A substitution in Lo18 did not prevent association with the membrane fraction, but it did, however, alter membrane fluidity regulation. A common characteristic of smHsp is the formation of oligomeric complexes. To evaluate the impact of the mutation on the multimeric organization of Lo18 produced by transformed E. coli, cross-linking experiments with formaldehyde were carried out, after which Western blotting confirmed multimeric structures (Fig. 5). As expected (Delmas et al., 2001), the cross-linking experiment revealed four major bands corresponding

to a monomer, a dimer, a trimer and a higher-order oligomer of the smHsp Lo18 WT. It is important to note that a second band observed below the Lo18 monomer corresponded to the truncated protein described previously (Coucheney et al., 2005). This result was obtained equally for the three proteins with amino acid substitutions. Recent studies indicate that smHsps have important biological functions in thermostability, disaggregation and proteolysis inhibition. Indeed, the understanding of these protein functions to enhance protein quality can be exploited for various applications such as nanobiotechnology, proteomics, bioproduction and bioseparation (Han et al., 2008). To apply prokaryotic smHsp in various biotechnological approaches, it is necessary to characterize the activity of such smHsp.