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 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.
Highlights
Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding
ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) is highly expressed in neurons and accounts for 1–2% of the brain proteome [30] and has been linked to neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) [31, 32]
For the 2/223 construct, the protein is attached to the DNA handles and pulled from its N and C termini, which should lead to the formation of a tightened 52 knot after mechanical unfolding
Summary
Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. Extensive experimental and computational studies have provided significant mechanistic insight into folding pathways [1,2,3,4,5] These proteins, which fold rapidly, have been shown to possess relatively smooth energy landscapes [6, 7]. Relatively few experimental studies on the knotting and folding of topologically knotted proteins have been published [14]. Most of these investigations have been focused on the trefoilknotted bacterial methyltransferases YibK and YbeA [16,17,18,19,20,21,22,23,24]. A detailed kinetic analysis undertaken by Luo et al has established that UCH-L1 exhibits
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