Knotting and unknotting of a protein in single molecule experiments

Abstract

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

How to fold intricately: using theory and experiments to unravel the properties of knotted proteins

“…Recently, optical tweezers were used on UCH-L1 to take it to three different unfolded states: unknotted, 3 1 -and 5 2 -knotted and refolding from such states was measured [61]. This study showed that threading to form either a 3 1 -or 5 2 -knotted state slowed folding as expected.…”

Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome

“…Their unique domain architecture – a single domain that binds to and hydrolyses the isopeptide bond of ubiqutinated substrates – with very slow off-rates for releasing hydrolysed ubiquitin products39 potentially increase the likelihood of encountering the proteasome machinery that actively unfolds ubiquitinated substrates for degradation. Indeed, it has been suggested by the recent single molecule mechanical unfolding study of UCH-L1 that the mechanical stability of the UCH domain may be beneficial for resisting UPS degradation40. Another important finding from the single molecule study is the consistent timescale of refolding triggered from mechanically and chemically unfolded states, implying that the intrinsic unfolding rates in the absence of chemical denaturant can be reliably derived from extrapolation of the linear dependency of chemical denaturant concentration (Fig.…”

Sequence and structural patterns detected in entangled proteins reveal the importance of co-translational folding

“…In either case, a simple protocol could consist in mutating into cysteine both residues at the ends of an entangled loop, provided no other cysteines are present in the sequence, and in assessing whether the folding is then hindered by the formation of a disulfide bridge in oxidizing conditions. In the context of knotted proteins, single molecule force spectroscopy techniques were shown to be particularly useful in controlling the topology of the unfolded state [46]. Similarly, both "in vivo" folding experiments [47] and appropriate simulation protocols [48][49][50] could be employed to test the possible role of cotranslational folding in determining the patterns detected for entangled motifs: double cysteine mutants would then be predicted to be more deleterious for the folding of C-terminal threads with respect to N-terminal threads.…”