Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments

Abstract

Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of crossdomain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.misfolding | off-pathway | rough energy landscape | optical tweezers L arge protein machines consist of long amino acid chains, often exceeding many hundreds or even over a thousand residues in length. Although the in vitro folding of small and mediumsized proteins is relatively well understood (1-5), very limited information exists about the complete folding process of such large proteins (6). In general, larger proteins often exhibit a multitude of intermediate and aggregation-prone misfolded states (4, 7). Recently, it has been shown that in multidomain proteins with homologous domains, cross-repeat intermediates can greatly slow down productive folding (8) but little is known about how size effects influence the folding of very large (>500 residues) nonhomologous multidomain proteins.Methods providing dynamic as well as structural information are rare, and many bulk methods often do not provide enough resolution to deal with the multitude of states expected for complex systems such as the aforementioned large protein complexes. Single-molecule force spectroscopy offers kinetic, energetic as well as coarse primary structural information combined with the possibility of actively manipulating systems, making it ideally suited for studying the folding of large proteins (5,(9)(10)(11)(12).In this paper, we study the folding and assembly of the large chaperone machinery heat shock protein 90 from yeast (Hsp90), a protein that needs to fold and self-assemble before it can function as a chaperone in the cell. Hsp90 consists of three domains, the N-terminal domain (N domain, 211 residues), the middle domain (M domain, 266 residues), and the C-terminal domain (C domain, 172 residues). In eukaryotic Hsp90, the N and M domains are connecte...

The ribosome destabilizes native and non‐native structures in a nascent multidomain protein

“…(D)] supports this interpretation. Similar effects have been observed for heat shock protein 90, another multidomain protein. In vivo , domain‐wise folding during synthesis likely avoids a scenario in which all domains in a given polypeptide molecule are simultaneously unfolded.…”

Hsp90 Sensitivity to ADP Reveals Hidden Regulation Mechanisms

“…Extensive structural analysis of diverse Hsp90 homologs (including crystallography, SAXS, FRET and electron microscopy) has led to a working model of the Hsp90 ATPase cycle [9–23]. Under apo and ADP conditions the Hsp90 dimer adopts a conformationally heterogeneous, and catalytically inactive, ensemble of open configurations in which the N-terminal domains (NTDs) of each monomer are predominantly separated.…”

A simple DNA handle attachment method for single molecule mechanical manipulation experiments

“…A simple approach for coupling DNA handles to proteins is to introduce cysteine residues at the point of attachment, allowing covalent linkage using thiol chemistry. [17][18][19][20][21][22][23][24][25] Thiol coupling imposes limitations, however, because all the other reactive cysteines must be removed, which is not always possible, and it becomes increasingly difficult with larger proteins that have more cysteines. HaloTag has been used, but requires the addition of a large protein domain to the target, which can limit its utility.…”