Sequence-specific size, structure, and stability of tight protein knots.

Approximately 1% of known protein structures display knotted configurations in their native fold, but the function of these configurations is not understood. It has been speculated that the entanglement may inhibit mechanical protein unfolding or transport, e.g., as in cellular threading or translocation processes through narrow biological pores. Protein knot manipulation, e.g., knot tightening and localization, has become possible in single-molecule experiments. Here, we investigate tight peptide knot (TPK) characteristics in detail by pulling selected 3(1) and 4(1)-knotted peptides using all-atom molecular dynamics computer simulations. We find that the 3(1)- and 4(1)-TPK lengths are typically Deltal approximately 47+/- 4 A and 69 +/- 4 A, respectively, for a wide range of tensions (0.1 nN less, similarF less, similar 1.5 nN). The 4(1)-knot length is in agreement with recent atomic force microscopy pulling experiments. Calculated TPK radii of gyration point to a pore diameter of approximately 20 A, below which a translocated knotted protein might get stuck. TPK characteristics, however, may be sequence-specific: we find a different size and structural behavior in polyglycines, and, strikingly, a strong hydrogen bonding and water trapping capability of hydrophobic TPKs. Water capture and release is found to be controllable by the tightening force in a few cases. These mechanisms result in a sequence-specific "locking" and metastability of TPKs, which might lead to a blocking of knotted peptide transport at designated sequence positions. We observe that macroscopic tight 4(1)-knot structures are reproduced microscopically ("figure of eight" versus the "pretzel") and can be tuned by sequence, in contrast to mathematical predictions. Our findings may explain a function of knots in native proteins, challenge previous studies on macromolecular knots, and prove useful in bio- and nanotechnology.