The translocation of polynucleotides through transmembrane protein pores is a fundamental biological process with important technological and medical relevance. The translocation process is complex, and it is influenced by a range of factors including the diameter and inner surface of the pore, the secondary structure of the polymer, and the interactions between the polymer and protein. In this paper, we perform nonequilibrium constant velocity-steered molecular dynamics simulations of nucleic acid molecule translocation through the protein nanopore α-hemolysin and use Jarzynski's identity to determine the associated free energy profiles. With this approach we are able to explain the observed differences in experimental translocation time through the nanopore between polyadenosine and polydeoxycytidine. The translocation of polynucleotides and single nucleotides through α-hemolysin is investigated. These simulations are computationally intensive as they employ models with atomistic level resolution; in addition to their size, these systems are challenging to study due to the time scales of translocation of large asymmetric molecules. Our simulations provide insight into the role of the interactions between the nucleic acid molecules and the protein pore. Mutated protein pores provide confirmation of residue-specific interactions between nucleotides and the protein pore. By harnessing such molecular dynamics simulations, we gain new physicochemical insight into the translocation process.

The translocation of nucleotide molecules across biological and synthetic nanopores has attracted attention as a next generation technique for sequencing DNA. Computer simulations have the ability to provide atomistic-level insight into important states and processes, delivering a means to develop a fundamental understanding of the translocation event, for example, by extracting the free energy of the process. Even with current supercomputing facilities, the simulation of many-atom systems in fine detail is limited to shorter timescales than the real events they attempt to recreate. This imposes the need for enhanced simulation techniques that expand the scope of investigation in a given timeframe. There are numerous free energy calculation and translocation methodologies available, and it is by no means clear which method is best applied to a particular problem. This article explores the use of two popular free energy calculation methodologies in a nucleotide-nanopore translocation system, using the α-hemolysin nanopore. The first uses constant velocity-steered molecular dynamics (cv-SMD) in conjunction with Jarzynski's equality. The second applies an adaptive biasing force (ABF), which has not previously been applied to the nucleotide-nanpore system. The purpose of this study is to provide a comprehensive comparison of these methodologies, allowing for a detailed comparative assessment of the scientific merits, the computational cost, and the statistical quality of the data obtained from each technique. We find that the ABF method produces results that are closer to experimental measurements than those from cv-SMD, whereas the net errors are smaller for the same computational cost. © 2014 The Authors Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

We describe nanoscale protein pores modified with a single hyperbranched dendrimer molecule inside the channel lumen. Sulfhydryl-reactive polyamido amine (PAMAM) dendrimers of generations 2, 3 and 5 were synthesized, chemically characterized, and reacted with engineered cysteine residues in the transmembrane pore alpha-hemolysin. Successful coupling was monitored using an electrophoretic mobility shift assay. The results indicate that G2 and G3 but not G5 dendrimers permeated through the 2.9 nm cis entrance to couple inside the pore. The defined molecular weight cutoff for the passage of hyperbranched PAMAM polymers is in contrast to the less restricted accessibility of flexible linear poly(ethylene glycol) polymers of comparable hydrodynamic volume. Their higher compactness makes sulfhydryl-reactive PAMAM dendrimers promising research reagents to probe the structure of porous membrane proteins with wide internal diameters. The conductance properties of PAMAM-modified proteins pores were characterized with single-channel current recordings. A G3 dendrimer molecule in the channel lumen reduced the ionic current by 45%, indicating that the hyperbranched and positively charged polymer blocked the passage of ions through the pore. In line with expectations, a smaller and less dense G2 dendrimer led to a less pronounced current reduction of 25%. Comparisons to recordings of PEG-modified pores revealed striking dissimilarities, suggesting that differences in the structural dynamics of flexible linear polymers vs compact dendrimers can be observed at the single-molecule level. Current recordings also revealed that dendrimers functioned as ion-selectivity filters and molecular sieves for the controlled passage of molecules. The alteration of pore properties with charged and hyperbranched dendrimers is a new approach and might be extended to inorganic nanopores with applications in sensing and separation technology.

The voltage-driven passage of biological polymers through nanoscale pores is an analytically, technologically, and biologically relevant process. Despite various studies on homopolymer translocation there are still several open questions on the fundamental aspects of the pore transport. One of the most important unresolved issues revolves around the passage of biopolymers which vary in charge and volume along their sequence. Here we exploit an experimentally tunable system to disentangle and quantify electrostatic and steric factors. This new, fundamental framework facilitates the understanding of how complex biopolymers are transported through confined space and indicates how biopolymer translocation can be slowed down to enable future sensing methods.SYNOPSIS: The voltage-driven translocation of complex biopolymers through a protein nanopore is biophysically modeled to disentangle and quantify electrostatic and steric factors which can oppose electrophoretic movement.

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