Sequence-Specific Size, Structure, and Stability of Tight Protein Knots

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

Section: Discussionsupporting

confidence: 77%

Knotting and unknotting of a protein in single molecule experiments

Ziegler

Lim

Mandal

et al. 2016

“…In these proteins, the disadvantage of less efficient folding may be balanced by a functional advantage connected with the presence of these knots. Numerous experimental (13)(14)(15)(16) and theoretical (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) studies have been devoted to understanding the precise nature of the structural and functional advantages created by the presence of these knots in protein backbones. It has been proposed that in some cases the protein knots and slipknots provide a stabilizing function that can act by holding together certain protein domains (4).…”

mentioning

confidence: 99%

Conservation of complex knotting and slipknotting patterns in proteins

Sułkowska

Rawdon

Millett

et al. 2012

228

229

While analyzing all available protein structures for the presence of knots and slipknots, we detected a strict conservation of complex knotting patterns within and between several protein families despite their large sequence divergence. Because protein folding pathways leading to knotted native protein structures are slower and less efficient than those leading to unknotted proteins with similar size and sequence, the strict conservation of the knotting patterns indicates an important physiological role of knots and slipknots in these proteins. Although little is known about the functional role of knots, recent studies have demonstrated a proteinstabilizing ability of knots and slipknots. Some of the conserved knotting patterns occur in proteins forming transmembrane channels where the slipknot loop seems to strap together the transmembrane helices forming the channel.protein knots | knot theory | topology T here are increasing numbers of known proteins that form linear open knots in their native folded structure (1, 2). In general, knots in proteins are orders of magnitude less frequent than would be expected for random polymers with similar length, compactness, and flexibility (3). In principle, the polypeptide chains folding into knotted native protein structures encounter more kinetic difficulties than unknotted proteins (4-12). Therefore, it is believed that knotted protein structures were, in part, eliminated during evolution because proteins that fold slowly and/or nonreproducibly should be evolutionarily disadvantageous for the hosting organisms. Nevertheless, there are several families of proteins that reproducibly form simple knots, complex knots, and slipknots (1, 2). In these proteins, the disadvantage of less efficient folding may be balanced by a functional advantage connected with the presence of these knots. Numerous experimental (13-16) and theoretical (17-27) studies have been devoted to understanding the precise nature of the structural and functional advantages created by the presence of these knots in protein backbones. It has been proposed that in some cases the protein knots and slipknots provide a stabilizing function that can act by holding together certain protein domains (4). In the majority of cases, however, one is unable to determine the precise structural and functional advantages provided by the presence of knots.To shed light on the function and formation of protein knots, we performed a thorough characterization of knotting within protein structures deposited in the Protein Data Bank (PDB) (28) by creating a precise mapping of the position and size of the knotted and slipknotted domains and knot tails (1, 2). We identified the types of knots that are formed by the backbone of the entire polypeptide chain and also by all continuous backbone portions of a given protein (1, 2). To characterize the knotting of proteins with linear backbones, one needs to set aside the orthodox rule of knot theory that states that all linear chains are unknotted because, by a continuous deformation,...

“…Trefoil, figure-of-eight, and penta knots with three, four, and five projected crossings of the polypeptide backbone, respectively, have been observed in proteins from all three domains of life (11)(12)(13). The functional advantages of knotted structures over their unknotted counterparts are unknown, although various computational studies have suggested that topological knots may increase protein stability or resistance to cellular translocation and degradation pathways (11,(14)(15)(16). Whereas various routes for protein knotting have been proposed, examples of which include the existence of an intermediate "slipknot" configuration or specific, attractive nonnative contacts that promote a threading event (17)(18)(19), these models have yet to be experimentally verified.…”

mentioning

confidence: 99%

Experimental detection of knotted conformations in denatured proteins

Mallam

Rogers

Jackson

2010

120

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.