Reactive derivatives of gramicidin enable light- and ion-modulated ion channels

Abstract

Detection of chemical processes on a single molecule scale is the ultimate goal of sensitive analytical assays. We have explored methods to detect chemical analytes in solution using synthetic derivatives of gramicidin A (gA). We exploited the functional properties of an ion channel-forming peptide-gA-to report changes in the local environment near the opening of these semi-synthetic nanopores upon exposure to specific external stimuli. These peptide-based nanosensors detect reaction-induced changes in the chemical or physical properties of functional groups presented at the opening of the pore. This paper discusses the development of gA-based sensors for detecting external factors such as metal ions in solution or for detecting specific wavelengths of light. We propose that gA-based ion channel sensors offer tremendous potential for ultra sensitive functional detection since a single chemical modification of each individual sensing element can lead to readily detectable changes in channel conductance.

A Semi-Synthetic Ion Channel Platform for Detection of Phosphatase and Protease Activity

“…We hypothesized that LF-catalyzed, proteolytic removal of four positive charges from this gA derivative (6) would yield a product with significantly increased single-channel conductance since the resulting product contained only one instead of four positive charges close to the entrance of the gA pore. We, 30,38,53 and others, 68,86 demonstrated in previous work that positive charges near the entrance of a gA pore reduce the single channel conductance of these pores due to electrostatic repulsion of the monovalent cations that are transported through gA pores. This effect is particularly pronounced in electrolyte solutions with low ionic strength 30 as used in the work presented here.…”

“…Permanent pores, such as α-hemolysin, do not provide this advantage because of the absence of complete opening and closing steps. Additionally, since gA reversibly partitions between the membrane and the bulk electrolyte solution, sensors based on a gA platform − can participate in homogeneous processes occurring in the bulk solution, whereas permanently incorporated pores are limited to heterogeneous processes on a membrane surface . Furthermore, gA is particularly well-suited for the development of an ion channel-based assay to detect enzyme activity because it is available in gram-scale quantities and conducive to synthetic derivatization while preserving its ion channel function; ,− it can be tailored for the detection of specific chemically or biochemically reactive analytes, ,,,,,,− and it is simple to use due to its spontaneous self-incorporation into bilayers and its well-defined, discrete ion conductance values.…”

Applications of biological pores in nanomedicine, sensing, and nanoelectronics

“…Additional approaches to manipulate the conductance of gramicidin pores by light consist of: (i) light-induced changes of the dipole moment within the pore lumen; [337] (ii) controlling the distance of a covalently attached channel-blocking group from the entrance of the pore [338]; or (iii) changing the charge of functional groups presented near the opening of the pore [192]. Two recent reviews highlighted further examples for the design and implementation of light-gated ion channels [246,334].…”

Engineered Ion Channels as Emerging Tools for Chemical Biology

“…First attempts at making light-switchable gramicidin pores have been reported. 48 Alternatively, ion channel proteins may be genetically engineered to respond to bio-orthogonal stimuli. One of the most dramatic approaches may be to use the recently introduced DNA origami pores combined with engineering stimuli responsive domains in this entirely new class of computer designed, DNA-based ion channels.…”