Alternative modes of client binding enable functional plasticity of Hsp70.

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

The Anti-Aggregation Holdase Hsp33 Promotes the Formation of Folded Protein Structures.

Holdase chaperones are known to be central to suppressing aggregation, but how they affect substrate conformations remains poorly understood. Here, we use optical tweezers to study how the holdase Hsp33 alters folding transitions within single maltose binding proteins and aggregation transitions between maltose binding protein substrates. Surprisingly, we find that Hsp33 not only suppresses aggregation but also guides the folding process. Two modes of action underlie these effects. First, Hsp33 binds unfolded chains, which suppresses aggregation between substrates and folding transitions within substrates. Second, Hsp33 binding promotes substrate states in which most of the chain is folded and modifies their structure, possibly by intercalating its intrinsically disordered regions. A statistical ensemble model shows how Hsp33 function results from the competition between these two contrasting effects. Our findings reveal an unexpectedly comprehensive functional repertoire for Hsp33 that may be more prevalent among holdases and dispels the notion of a strict chaperone hierarchy.

Trehalose facilitates DNA melting: a single-molecule optical tweezers study.

Using optical tweezers, here we show that the overstretching transition force of double-stranded DNA (dsDNA) is lowered significantly by the addition of the disaccharide trehalose as well as certain polyol osmolytes. This effect is found to depend linearly on the logarithm of the trehalose concentration. We propose an entropic driving mechanism for the experimentally observed destabilization of dsDNA that is rooted in the higher affinity of the DNA bases for trehalose than for water, which promotes base exposure and DNA melting. Molecular dynamics simulation reveals the direct interaction of trehalose with nucleobases. Experiments with other osmolytes confirm that the extent of dsDNA destabilization is governed by the ratio between polar and apolar fractions of an osmolyte.