Effect of Hsp70 Chaperone on the Folding and Misfolding of Polypeptides Modeling an Elongating Protein Chain

Abstract

Virtually nothing is known about the interaction of co-translationally active chaperones with nascent polypeptides and the resulting effects on peptide conformation and folding. We have explored this issue by NMR analysis of apomyoglobin N-terminal fragments of increasing length, taken as models for different stages of protein biosynthesis, in the absence and presence of the substrate binding domain of Escherichia coli Hsp70, DnaK-β. The incomplete polypeptides misfold and self-associate under refolding conditions. In the presence of DnaK-β, however, formation of the original selfassociated species is completely or partially prevented. Chaperone interaction with incomplete protein chains promotes a globally unfolded dynamic DnaK-β-bound state, which becomes foldingcompetent only upon incorporation of the residues corresponding to the C-terminal H helix. The chaperone does not bind the full-length protein at equilibrium. However, its presence strongly disfavors the kinetic accessibility of misfolding side-routes available to the full-length chain. This work supports the role of DnaK as a "holder" for incomplete N-terminal polypeptides. However, as the chain approaches its full-length status, the tendency to intramolecularly bury non-polar surface efficiently out-competes chaperone binding. Under these conditions, DnaK serves as a "folding enhancer" by supporting folding of a population of otherwise folding-incompetent full-length protein chains.

Binding Specificity of an α-Helical Protein Sequence to a Full-Length Hsp70 Chaperone and Its Minimal Substrate-Binding Domain

“…The residues corresponding apoMb's binding sites for DnaK may be exposed during protein biosynthesis, upon apoMb expression in E. coli (routinely performed during the production of this protein for biophysical studies), when the chain is incomplete and cannot properly fold. In agreement with the above, purified N-terminal fragments of apoMb interact with DnaK-, while the full-length protein does not, at equilibrium (11). It is interesting to note that these N-terminal fragments selfassociate and adopt non-native -sheet structure, when taken out of the context of the native protein (19).…”

“…Its typical globin fold is extensively represented across all three kingdoms of life (13). Sperm whale apoMb is the best characterized member of the globin family, in terms of its structure, folding mechanisms, and interaction with chaperones (11,(14)(15)(16)(17)(18)(19). This protein is routinely produced in bacterial expression systems (20,21), and it is a suitable model for investigating substrate interactions with chaperones, because portions of its sequence are known to severely self-associate in the absence of chaperones (19).…”

Conformational dynamics of the molecular chaperone Hsp90

“…Hsp60 (GroEL) and Hsp70 (DnaK) represent two well studied members of this class of proteins. Both Hsp60 and Hsp70 interact with nascent polypeptide chains and promote their folding by interacting with hydrophobic surfaces on substrate proteins (Gomez-Puertas et al, 2004;Kurt et al, 2006;Rudiger et al, 1997;Weissman et al, 1995). Once substrates are bound, the chaperone undergoes subsequent rounds of ATP hydrolysis causing conformational changes within the chaperone (Young et al, 2004).…”

Hsp70 biases the folding pathways of client proteins

“…In contrast to the detailed structural studies characterizing DnaK, little atomic resolution data are available on the conformation of folding-competent client proteins in the DnaK-bound state. It is known that DnaK binds substrates in a globally unfolded conformation with varying degrees of local residual native and nonnative secondary structure (9)(10)(11)(12)(13). However, whether stable or transient long-range interactions are present in DnaK-bound client proteins remains an open question that has relevance for understanding the function of DnaK in substrate refolding and disaggregation.…”