Imbalances in the eye lens proteome are linked to cataract formation.

The prevalent model for cataract formation in the eye lens posits that damaged crystallin proteins form light-scattering aggregates. The α-crystallins are thought to counteract this process as chaperones by sequestering misfolded crystallin proteins. In this scenario, chaperone pool depletion would result in lens opacification. Here we analyze lenses from different mouse strains that develop early-onset cataract due to point mutations in α-, ß-, or γ-crystallin proteins. We find that these mutant crystallins are unstable in vitro; in the lens, their levels are substantially reduced, and they do not accumulate in the water-insoluble fraction. Instead, all the other crystallin proteins, including the α-crystallins, are found to precipitate. The changes in protein composition and spatial organization of the crystallins observed in the mutant lenses suggest that the imbalance in the lenticular proteome and altered crystallin interactions are the bases for cataract formation, rather than the aggregation propensity of the mutant crystallins.

Knotting and unknotting of a protein in single molecule experiments.

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.

Molecular knots in biology and chemistry.

Knots and entanglements are ubiquitous. Beyond their aesthetic appeal, these fascinating topological entities can be either useful or cumbersome. In recent decades, the importance and prevalence of molecular knots have been increasingly recognised by scientists from different disciplines. In this review, we provide an overview on the various molecular knots found in naturally occurring biological systems (DNA, RNA and proteins), and those created by synthetic chemists. We discuss the current knowledge in these fields, including recent developments in experimental and, in some cases, computational studies which are beginning to shed light into the complex interplay between the structure, formation and properties of these topologically intricate molecules.

Mechanistic insights into the folding of knotted proteins in vitro and in vivo.

The importance of knots and entanglements in biological systems is increasingly being realized and the number of proteins with topologically complex knotted structures has risen. However, the mechanism as to how these proteins knot and fold efficiently remains unclear. Using a cell-free expression system and pulse-proteolysis experiments, we have investigated the mechanism of knotting and folding for two bacterial trefoil-knotted methyltransferases. This study provides the first experimental evidence for a knotting mechanism. Results on fusions of stable protein domains to N-terminus, C-terminus or both termini of the knotted proteins clearly demonstrate that threading of the nascent chain through a knotting loop occurs via the C-terminus. Our results strongly suggest that this mechanism occurs even when the C-terminus is severely hindered by the addition of a large stable structure, in contrast to some simulations indicating that even the folding pathways of knotted proteins have some plasticity. The same strategy was employed to probe the effects of GroEL-GroES. In this case, results suggest active mechanisms for the molecular chaperonin. We demonstrate that a simple model in which GroEL-GroES sterically confines the unknotted polypeptide chain thereby promoting knotting is unlikely, and we propose two alternatives: (a) the chaperonin facilitates unfolding of kinetically and topologically trapped intermediates or (b) the chaperonin stabilizes interactions that promote knotting. These findings provide mechanistic insights into the folding of knotted proteins both in vitro and in vivo, thus elucidating how they have withstood evolutionary pressures regardless of their complex topologies and intrinsically slow folding rates.