Information Encoded in Non-Native States Drives Substrate-Chaperone Pairing

Abstract

Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones.

Monitoring conformational heterogeneity of the lid of DnaK substrate‐binding domain during its chaperone cycle

“…This led us to investigate the conformational states of the SBD in terms of its lid‐base distance after binding to the cellular substrates in their non‐native states, the state in which intracellular chaperone–substrate pairing takes place. We took a panel of E. coli proteins that has already been demonstrated to interact with DnaK/J chaperones (DM‐MBP) or are expected to bind to these chaperone machineries (known GroEL/ES substrates which are thought to get transferred from upstream DnaK/J system) like MetK, Ths or DapA . These substrate proteins are of varied size and structure (30–60 kDa) and slowly refold during in vitro refolding reactions upon dilution from denaturant and populate kinetically stable intermediates.…”

“…In the past decade, single‐molecule FRET has been established as a highly efficient methodology to decipher conformational heterogeneity and molecular dynamics of bio‐molecules at single‐molecule resolution. In earlier studies, we and others have extensively employed sm‐FRET to decipher the conformational heterogeneity of various chaperones and its substrates within in vitro and ex vivo conditions . In the present study, we have employed a set of FRET‐based sensors that efficiently report for intradomain conformational changes of the SBD of DnaK due to movement of the alpha helical lid away from the base of the substrate‐binding domain during DnaK functional cycle.…”

Large-Scale Evolutionary Analyses on SecB Subunits of Bacterial Sec System

“…Indeed, it was showed that cotranslational substrate recognition by SecB was greatly suppressed in the presence of ribosome-bound trigger factor, but not by DnaK [82]. Still, it was suggested that the general evolution of chaperones would favor the sequences that coded both the functional native state and folding intermediate with high affinity as well [87]. Another possible factor, which affects the evolution of SecB, would be the Tat system, which transfers folded secreted proteins and plays a complementary role with SecB [88].…”

Atypical domain communication and domain functions of a Hsp110 chaperone

“…We made three such pairs to monitor the changes in distance due to possible conformational alterations: 1. between NBD and SBD (E319C-D412C), 2. between the lid and base of SBD (D412C-D600C) and 3. between the SBD-lid and NBD (K45C-K600C) ( Figure 1A). We took single molecule FRET measurements of donor-acceptor labelled Sse1 proteins in solution in extremely low concentration (~50pM) to achieve the single molecule resolution as described before (Mapa, Tiwari et al, 2012).…”