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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 52- 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 31 or 52 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 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot. [ABSTRACT FROM AUTHOR]

Structures that contain a knot formed by the path of the polypeptide backbone represent some of the most complex topologies observed in proteins. How or why these topological knots arise remains unclear. By developing a method to experimentally trap and detect knots in nonnative polypeptide chains, we find that two knotted methyltransferases, YibK and YbeA, can exist in a trefoil-knot conformation even in their chemically unfolded states. The unique denatured-state topology of these molecules explains their ability to efficiently fold to their native knotted structures in vitro and offers insights into the potential role of knots in proteins. Furthermore, the high prevalence of the denatured-state knots identified here suggests that they are either difficult to untie or that threading of any untied molecules is rapid and spontaneous. The occurrence of such knotted topologies in unfolded polypeptide chains raises the possibility that they could play an important, and as yet unexplored, role in folding and misfolding processes in vivo. [ABSTRACT FROM AUTHOR]

One of the most striking topological features to be found in a protein is that of a distinct knot formed by the path of the polypeptide backbone. Such knotted structures represent some of the smallest "self-tying" knots observed in Nature. Proteins containing a knot deep within their structure add an extra complication to the already challenging protein-folding problem; it is not obvious how, during the process of folding, a substantial length of polypeptide chain manages to spontaneously thread itself through a loop. Here, we probe the folding mechanism of YibK, a homodimeric α/β-knot protein containing a deep trefoil knot at its carboxy terminus. By analyzing the effect of mutations made in the knotted region of the protein we show that the native structure in this area remains undeveloped until very late in the folding reaction. Single-site destabilizing mutations made in the knot structure significantly affect only the folding kinetics of a late-forming intermediate and the slow dimerization step. Furthermore, we find evidence to suggest that the heterogeneity observed in the denatured state is not caused by isomerization of the single cis proline bond as previously thought, but instead could be a result of the knotting mechanism. These results allow us to propose a folding model for YibK where the threading of the polypeptide chain and the formation of native structure in the knotted region of the protein occur independently as successive events. [ABSTRACT FROM AUTHOR]

Getting high-resolution structures of large proteins in solution has always been a challenge. In this issue of Structure, Krukenberg et al. have used new methods of analyzing SAXS data to reveal a novel conformation of Hsp90 in solution. [Copyright &y& Elsevier]

Summary: Proteins that contain a distinct knot in their native structure are impressive examples of biological self-organization. Although this topological complexity does not appear to cause a folding problem, the mechanisms by which such knotted proteins form are unknown. We found that the fusion of an additional protein domain to either the amino terminus, the carboxy terminus, or to both termini of two small knotted proteins did not affect their ability to knot. The multidomain constructs remained able to fold to structures previously thought unfeasible, some representing the deepest protein knots known. By examining the folding kinetics of these fusion proteins, we found evidence to suggest that knotting is not rate limiting during folding, but instead occurs in a denatured-like state. These studies offer experimental insights into when knot formation occurs in natural proteins and demonstrate that early folding events can lead to diverse and sometimes unexpected protein topologies. [Copyright &y& Elsevier]