Marcus-like translocation kinetics of a knotted protein.

Abstract

It has been known that some proteins naturally form knots, wherein the amino acid chain is tied up in the protein in its functional form. However, much less is known about the transport kinetics of such knotted proteins as these are forced to traverse ultrasmall pores using an electric field. Previous computational studies indicate that the translocation of knotted protein can be blocked. Recent nanopore experiments demonstrated that localized electrical fields in the vicinity of a nanopore could drive the translocation coupled to the partial or complete unfolding of unknotted proteins without requiring chemical denaturants, enzymes, or a physical pulling force. Here we combine single-molecule experiments and all-atom MD simulations to study the electrical unfolding behavior of two knotted proteins, YibK and YbeA, for pore diameters (dpore) ranging from 1.5-7 nm. We found that YibK is ∼15 kBT more stable than YbeA, and that both proteins unfold through several distinct intermediate states and consist of two distinct denatured configurations. For pores larger than 3 nm diameters, the more stable YibK translocates faster than YbeA. In contrast, for pores below 3 nm in diameter, we found no evidence of translocation for YibK, and further, YbeA can translocate even for pore diameters below 2 nm. These observations can be explained by the conformation-translocation coupling mechanism which suggests that access to an intermediate and unfolded state is critical for the translocation (for dpore< dprotein). This can be achieved by controlling the external electric field without the need for an enzyme or a large mechanical pulling force. In the experiments with YbeA using pores having a diameter range of 3-4 nm, we observed that the translocation rate first increases by increasing voltage and then starts decreasing above a threshold voltage, suggesting Marcus-like translocation kinetics behavior.