Electroosmotic flow (EOF) is the primary driving force behind protein translocation in nanopore systems. Molecular dynamics (MD) simulations revealed that guanidium (Gdm + ) ions stick to negatively charged and hydrophobic residues, creating a nanopore with a positively charged interior that attracts negatively charged molecules, such as Cl - , which have a large hydration shell. This shell drags water molecules, generating an EOF under an applied voltage which pulls an unfolded protein through the pore. CytK is a biological nanopore that bears a large constriction site to allow for the passage of unfolded proteins and a higher signal-to-noise ratio compared to other commonly used pores. Although GdmCl increases the electroosmotic flow in CytK, high concentrations of this salt can affect the integrity of the nanopore, membrane, and substrates, so it is important that a high level of EOF does not depend entirely on GdmCl concentrations. This study aims to create an intrinsically anion-selective CytK nanopore to reduce Gdm + dependence for generating an EOF. Rosetta is a protein modeling software that we used to mutate the residues of the interior surface of the CytK β barrel into charged and polar residues. Seven mutants were selected for expression in the BL21(DE3) strain of E. coli as inclusion bodies: T118K, Q147K, T118K-Q147K, Q147K-Q124E, Q147K-Q124S, Q147K-Q124E-T118K, and Q147K-Q124S-T118K. We are currently characterizing these mutant variants’ ability to increase the positive charge of the CytK pore walls and to enhance Cl − selectivity and electroosmotic flow. We evaluate the enhancements in protein translocation rates, as well as the signal obtained during fingerprinting of proteins and their isoforms.
Mandujano et al. (Sun,) studied this question.