Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 5 2 -knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 3 1 or 5 2 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 3 1 knot, we measure a significantly larger size for the 5 2 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 5 2 knot compared with a 3 1 knot.knotted proteins | protein folding | single molecule | optical tweezers | ubiquitin C-terminal hydrolase

No abstract

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α-and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.laser trapping | parallel pathways | elasticity | force | protein extension W hen looking at protein structures at atomic resolution, it is often tempting to use macroscopic mechanical analogies to describe their function as molecular machines. However, such analogies are often misleading because boundaries between independently stable subdomains cannot often be determined from structures, owing to the high cooperativity of protein folding and structural transitions. Single-molecule protein nanomechanics have emerged as a tool to force biomolecules through their conformational space and, hence, identify hinges, breaking points, and mechanically stable subdomains (1-3).A prominent example of a protein machine undergoing large conformational change during its functional cycle is the ATP-regulated Hsp70 chaperone DnaK-a central molecular chaperone of the protein quality control network in a cell (4-6). Once ATP is bound to the nucleotide binding domain (NBD, blue-yellow; Fig. 1A) of DnaK, the initially closed substrate binding domain (SBD) opens its binding cleft by engaging the β-subdomain to the NBD. In doing so, it undergoes a dramatic ∼10 Å displacement of its lid subdomain ( Fig. 1A; refs. 7 and 8) to allow exchange of substrates (9). Several crystal structures of the isolated SBD (in which the NBD is absent) have been solved (10-15). In these structures, the absence or presence of peptide clients or nonnatural ligands induce no significant structural changes in the closed conformation. There is no indication in the crystal structures of the huge conformational change of the lid domain of the SBD, seen in the ATP form of the full-length two-domain DnaK. Therefore, the large conformational change of the SBD is only observed in the two-domain DnaK after ATP binding. Thus, although the crystal structures provide us with valuable insights into the 3D arrangement of individual atoms, the thermodynamic and mechanical stability of individual substructures are difficult to predict based on this information alone. Here, we ask how the large ATP-induced changes of the SBD, as seen in the two-domain DnaK, are mirrored in the subdomain integrity and nano...

Biomolecules such as nucleic acids and proteins constitute the cells and its organelles that form the crucial components in all living organisms. They are associated with a variety of cellular processes during which they undergo conformational orientations. The structural rearrangements resulting from protein−protein, protein− DNA, and protein−drug interactions vary in spatial and temporal length scales. Force is one of the important key factors which regulate these interactions. The magnitude of the force can vary from sub-piconewtons to several thousands of piconewtons. Singlemolecule force spectroscopy acts as a powerful tool which is capable of investigating mechanical stability and conformational rearrangements arising in biomolecules due to the above interactions. Real-time observation of conformational dynamics including access to rare or transient states and the estimation of mean dwell times using these tools aids in the kinetic analysis of these interactions. In this review, we highlight the capabilities of common force spectroscopy techniques such as optical tweezers, magnetic tweezers, and atomic force microscopy with case studies on emerging applications.

No abstract