Tying up the Loose Ends: A Mathematically Knotted Protein.

Knots have attracted scientists in mathematics, physics, biology, and engineering. Long flexible thin strings easily knot and tangle as experienced in our daily life. Similarly, long polymer chains inevitably tend to get trapped into knots.

Topologically knotted deubiquitinases exhibit unprecedented mechanostability to withstand the proteolysis by an AAA+ protease.

More than one thousand knotted protein structures have been identified so far, but the functional roles of these knots remain elusive. It has been postulated that backbone entanglement may provide additional mechanostability. Here, we employed a bacterial proteasome, ClpXP, to mechanically unfold 5-knotted human ubiquitin C-terminal hydrolase (UCH) paralogs from their C-termini, followed by processive translocation into the proteolytic chamber for degradation.

A Natively Monomeric Deubiquitinase UCH-L1 Forms Highly Dynamic but Defined Metastable Oligomeric Folding Intermediates.

Oligomerization of misfolded protein species is implicated in many human disorders. Here we showed by size-exclusion chromatography-coupled multiangle light scattering (SEC-MALS) and small-angle X-ray scattering (SEC-SAXS) that urea-induced folding intermediate of human ubiquitin C-terminal hydrolase, UCH-L1, can form well-defined dimers and tetramers under denaturing conditions despite being highly disordered. Introduction of a Parkinson disease-associated mutation, I93M, resulted in increased aggregation propensity and formation of irreversible precipitants in the presence of a moderate amount of urea.

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

Human ubiquitin C-terminal hydrolyase UCH-L5 is a topologically knotted deubiquitinase that is activated upon binding to the proteasome subunit Rpn13. The length of its intrinsically disordered cross-over loop is essential for substrate recognition. Here, we showed that the catalytic domain of UCH-L5 exhibits higher equilibrium folding stability with an unfolding rate on the scale of 10 s, over four orders of magnitudes slower than its paralogs, namely UCH-L1 and -L3, which have shorter cross-over loops.

Random-Coil Behavior of Chemically Denatured Topologically Knotted Proteins Revealed by Small-Angle X-ray Scattering.

Recent studies on the mechanisms by which topologically knotted proteins attain their natively knotted structures have intrigued theoretical and experimental biophysicists. Of particular interest is the finding that YibK and YbeA, two small trefoil knotted proteins, remain topologically knotted in their chemically denatured states. Using small-angle X-ray scattering (SAXS), we examine whether these chemically denatured knotted proteins are different from typical random coils.