Matthias Rief

Hsp90 is an essential molecular chaperone, which has to be in a dimeric form for its correct function. While the affinity of the dimer has previously been measured, little is known about how it associates and dissociates and the factors that influence this. We perform an in-depth single molecule characterization of the C-terminal association and dissociation of Hsp90. We find more than one dissociation rate, indicating that the dimer has a stable and an unstable state. Furthermore, we find that the stability of the C-terminal association is dependent on the presence of ATP, despite the C-terminal dimerization interface being distal to the catalytic site.

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183–383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily. Significance Proteins with a similar structure can have largely different folding properties. Although some fold readily, others can only assume their native structure through the help of chaperone proteins. Partially folded intermediates play a key role in defining those folding differences. However, owing to their transient nature, they are not amenable to the structural investigation. Using a combination of single-molecule mechanics, protein engineering, and crystallography, we identified a stable native-like functional nucleus, which is a critical intermediate for spontaneous folding of the Hsp70 nucleotide-binding domain. Based on our findings, we engineered a chimera turning a homologous but folding-incompetent protein into a spontaneously folding protein that is enzymatically active. Our results have implications for the folding of actin from the same superfamily.

Significance: Green fluorescent protein (GFP) is widely used as a tag to watch otherwise invisible proteins and as a sensor of its local chemical environment. Since GFP can form many partially folded states, it is critical to know how these structural changes affect its signature fluorescence. We use optical tweezers to force single molecules of GFP into folding and unfolding intermediate states and simultaneously probe single-molecule fluorescence from each state. It was found that GFP fluorescence requires complete structural integrity; none of the unfolding or refolding intermediates were observed to fluoresce, but fluorescence could be recovered by complete refolding. This feature was exploited to reversibly, mechanically switch GFP between on and off fluorescence states.

Significance: Hsp70 is a protein nanometer-sized molecular machine operating similar like a macroscopic engine: Combustion of fuel (ATP in the case of Hsp70) triggers mechanical motion for its function. However, in contrast to macroscopic engines, protein machines face additional limitations imposed by thermal fluctuations and by the low stability of the individual components. Using single-molecule force spectroscopy, we drive the passive machine through its mechanical states that allows us to detect its relevant parts. Applying this approach for the substrate binding domain (SBD) of Hsp70, we identified mechanical subunits and hinges of the SBD. Interestingly, these parts are involved in an ATP-fueled biological conformational change of Hsp70. In summary, single-molecule manipulation gives insight into the operating principles of nanoscale molecular machines.

Significance: Muscle is the tissue in our body experiencing most extreme mechanical forces. The mechanism of active force generation has been investigated for more than 50 y and is fairly well understood. However, despite its physiological significance, it is still unknown what mechanical linkages hold together the muscle machinery under passive stretching forces. In this paper, we show with direct mechanical single-molecule measurements that an array of titin/α-actinin bonds composes a dynamic network that can provide stable anchoring, maintaining the integrity of the muscle Z-disk even under load. This dynamic network explains how components of the Z-disk are able to rapidly rearrange and, at the same time, form a long-term stable mechanical structure.

Significance: Knots and entanglements are common in biology, but little is known about their formation or function. Here, we use state-of-the-art single-molecule force spectroscopy to mechanically unfold a protein, ubiquitin C-terminal hydrolase isoenzyme L1, which has a 5 ₂ knot in its structure. The exquisite control inherent in this technique enables us to specifically unfold the protein to different knotted and unknotted denatured states, from which we can then watch the protein refold. These experiments establish the effect of knots on folding pathways. Many intermediate structures are detected, suggesting both on- and off-pathway intermediates are populated. Our results also highlight the potential difficulties in degrading a 5 ₂ -knotted protein and therefore may have implications for some diseases.

Enzymes are molecular machines that bind substrates specifically, provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates. Direct information about the energetics that drive conformational changes is difficult to obtain. We used subnanometre single-molecule force spectroscopy to study the energetic drive of substrate-dependent lid closing in the enzyme adenylate kinase. Here we show that in the presence of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A), closing and opening of both lids is cooperative and tightly coupled to inhibitor binding. Surprisingly, binding of the substrates ADP and ATP exhibits a much smaller energetic drive towards the fully closed state. Instead, we observe a new dominant energetic minimum with both lids half closed. Our results, combining experiment and molecular dynamics simulations, give detailed mechanical insights into how an enzyme can cope with the seemingly contradictory requirements of rapid substrate exchange and tight closing, to ensure efficient catalysis.

Significance: Understanding protein folding is, as yet, an unsolved question in the life sciences that has relevance for many diseases. While the folding of simple and small protein domains is well studied, for large proteins, the abundance of pathways and intermediate states makes them difficult to characterize using standard protein folding experiments. With single-molecule optical tweezers experiments, we can overcome these limitations. We observe in real time the folding of a dimeric, three-domain protein from the fully unfolded chain to the biologically active, quaternary structure. The likelihood of the folding process being hindered by misfolded intermediates increases with chain length. These misfolded states can slow down folding significantly and may lead to aggregation in vivo.