Single-Molecule Detection Reveals Different Roles of Skp and SurA as Chaperones

Abstract

Skp and SurA are both periplasmic chaperones involved in the biogenesis of Escherichia coli β-barrel outer membrane proteins (OMPs). It is commonly assumed that SurA plays a major role whereas Skp is a minor factor. However, there is no molecular evidence for whether their roles are redundant. Here, by using different dilution methods, we obtained monodisperse and aggregated forms of OmpC and studied their interactions with Skp and SurA by single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We found that Skp can dissolve aggregated OmpC while SurA cannot convert aggregated OmpC into the monodisperse form and the conformations of OmpC recognized by the two chaperones as well as their stoichiometries of binding are different. Our study demonstrates the functional distinctions between Skp and SurA. In particular, the role of Skp is not redundant and is probably more significant under stress conditions.

High affinity of Skp to OmpC revealed by single-molecule detection

“…S13). The 0.78 peak was assigned to apo-OmpC and the 0.13 peak to bound-OmpC as previously observed 6 . The peak at Eapp=0 (zero peak) was due to missing or inactivated acceptors and was disregarded.…”

“…As OMPs are synthesized in cytoplasm in unfolded state and have to transport through aqueous periplasm to fold in OM, several periplasmic quality control factors such as survival protein A (SurA), seventeen kilodalton protein (Skp) and serine endoprotease DegP are involved to protect OMPs from mis-folding and aggregation [2][3][4][5] . Recent study from our group identifies Skp to be the major protein to prevent OMPs from aggregation 6 . Accumulation of periplasmic OMPs and other survival stress will stimulate the σ E response 7 , which downregulates OMPs expression and upregulates chaperone expression 8 .…”

“…To overcome above problems, subpopulation needs to be separately resolved by, for example, electrophoresis, chromatography, or single-molecule detection (SMD) 6,[21][22][23] . Unlike other separation methods which perturb the molecular interaction and the equilibrium of subpopulations, SMD yields a statistical analysis of individual molecule via ultrahigh spatiotemporal resolution.…”

“…Unlike other separation methods which perturb the molecular interaction and the equilibrium of subpopulations, SMD yields a statistical analysis of individual molecule via ultrahigh spatiotemporal resolution. Furthermore, SMD may prepare and investigate OMP samples under monomeric form, so that the interference of OMP aggregation can be completely removed 6,24 . Here we developed an easy-to-implement SMD method named the portion selectively chosen fluorescence correlation spectroscopy (pscFCS), which takes advantages of single-molecule fluorescence resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS) to get diffusion time of specific subpopulation.…”

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

“…The results presented here demonstrate a role for SurA interdomain dynamics in OMP binding, notably the reorganisation of the core and P1 domains, and dynamic localisation of P2 close to these domains. Such structural plasticity may also be important for facilitating binding of SurA:OMP complexes to BAM, assisting BAM catalytic activity, or priming the OMP for membrane insertion by pre-selecting favourable conformations for folding, thereby smoothing the energy landscape of folding 42,71,72 .…”