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1. Electrical unfolding of cytochrome c during translocation through a nanopore constriction

   https://doi.org/10.1073/pnas.2016262118  

   Tripathi, Prabhat; Benabbas, Abdelkrim; Mehrafrooz, Behzad; Yamazaki, Hirohito; Aksimentiev, Aleksei; Champion, Paul M.; Wanunu, Meni (April 2021, Proceedings of the National Academy of Sciences)

   null (Ed.)

   Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field–driven translocation behavior of cytochrome c (cyt c ) through ultrathin silicon nitride (SiN x ) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field–induced deformability. 

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2. Threading single proteins through pores to compare their energy landscapes

   https://doi.org/10.1073/pnas.2202779119  

   Tripathi, Prabhat; Firouzbakht, Arash; Gruebele, Martin; Wanunu, Meni (September 2022, Proceedings of the National Academy of Sciences)

   Translocation of proteins is correlated with structural fluctuations that access conformational states higher in free energy than the folded state. We use electric fields at the solid-state nanopore to control the relative free energy and occupancy of different protein conformational states at the single-molecule level. The change in occupancy of different protein conformations as a function of electric field gives rise to shifts in the measured distributions of ionic current blockades and residence times. We probe the statistics of the ionic current blockades and residence times for three mutants of the $\lambda$-repressor family in order to determine the number of accessible conformational states of each mutant and evaluate the ruggedness of their free energy landscapes. Translocation becomes faster at higher electric fields when additional flexible conformations are available for threading through the pore. At the same time, folding rates are not correlated with ease of translocation; a slow-folding mutant with a low-lying intermediate state translocates faster than a faster-folding two-state mutant. Such behavior allows us to distinguish among protein mutants by selecting for the degree of current blockade and residence time at the pore. Based on these findings, we present a simple free energy model that explains the complementary relationship between folding equilibrium constants and translocation rates. 

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3. Translocation Behaviors of Synthetic Polyelectrolytes through Alpha-Hemolysin (α-HL) and Mycobacterium smegmatis Porin A (MspA) Nanopores

   https://doi.org/10.1149/1945-7111/ac6c55  

   Wang, Xiaoqin; Stevens, Kaden C.; Ting, Jeffrey M.; Marras, Alexander E.; Rezvan, Gelareh; Wei, Xiaojun; Taheri-Qazvini, Nader; Tirrell, Matthew V.; Liu, Chang (May 2022, Journal of The Electrochemical Society)

   DNAs have been used as probes for nanopore sensing of noncharged biomacromolecules due to its negative phosphate backbone. Inspired by this, we explored the potential of diblock synthetic polyelectrolytes as more flexible and inexpensive nanopore sensing probes by investigating translocation behaviors of PEO-b-PSS and PEO-b-PVBTMA through commonly used alpha-hemolysin ( α -HL) and Mycobacterium smegmatis porin A (MspA) nanopores. Translocation recordings in different configurations of pore orientation and testing voltage indicated efficient PEO-b-PSS translocations through α -HL and PEO-b-PVBTMA translocations through MspA. This work provides insight into synthetic polyelectrolyte-based probes to expand probe selection and flexibility for nanopore sensing. 

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4. Laser-based temperature control to study the roles of entropy and enthalpy in polymer-nanopore interactions

   https://doi.org/10.1126/sciadv.abf5462  

   Angevine, Christopher E.; Robertson, Joseph W.F.; Dass, Amala; Reiner, Joseph E. (April 2021, Science Advances)

   null (Ed.)

   Single-molecule approaches for probing the free energy of confinement for polymers in a nanopore environment are critical for the development of nanopore biosensors. We developed a laser-based nanopore heating approach to monitor the free energy profiles of such a single-molecule sensor. Using this approach, we measure the free energy profiles of two distinct polymers, polyethylene glycol and water-soluble peptides, as they interact with the nanopore sensor. Polyethylene glycol demonstrates a retention mechanism dominated by entropy with little sign of interaction with the pore, while peptides show an enthalpic mechanism, which can be attributed to physisorption to the nanopore (e.g., hydrogen bonding). To manipulate the energetics, we introduced thiolate-capped gold clusters [Au 25 (SG) 18 ] into the pore, which increases the charge and leads to additional electrostatic interactions that help dissect the contribution that enthalpy and entropy make in this modified environment. These observations provide a benchmark for optimization of single-molecule nanopore sensors. 

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5. Membrane-selective nanoscale pores in liposomes by a synthetically evolved peptide: implications for triggered release

   https://doi.org/10.1039/d1nr03084a  

   Sun, Leisheng; Hristova, Kalina; Wimley, William C. (July 2021, Nanoscale)

   null (Ed.)

   Peptides that form nanoscale pores in lipid bilayers have potential applications in triggered release, but only if their selectivity for target synthetic membranes over bystander biomembranes can be optimized. Previously, we identified a novel family of α-helical pore-forming peptides called “macrolittins”, which release macromolecular cargoes from phosphatidylcholine (PC) liposomes at concentrations as low as 1 peptide per 1000 lipids. In this work, we show that macrolittins have no measurable cytolytic activity against multiple human cell types even at high peptide concentration. This unprecedented selectivity for PC liposomes over cell plasma membranes is explained, in part, by the sensitivity of macrolittin activity to physical chemical properties of the bilayer hydrocarbon core. In the presence of cells, macrolittins release all vesicle-entrapped cargoes (proteins and small molecule drugs) which are then readily uptaken by cells. Triggered release occurs without any direct effect of the peptide on the cells, and without vesicle–vesicle or vesicle–cell interactions. 

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