Aerolysin, a Powerful Protein Sensor for Fundamental Studies and Development of Upcoming Applications

Abstract

Nanopore electrical approach is a breakthrough in single molecular level detection of particles as small as ions, and complex as biomolecules. This technique can be used for molecule analysis, and characterization as well as for the understanding of confined medium dynamics in chemical or biological reactions. Altogether, the information obtained from these kinds of experiments will allow to address challenges in a variety of biological fields. The sensing, design and manufacture of nanopores is crucial to obtain these objectives. For some time now, aerolysin, a pore forming toxin, and its mutants have shown high potential in real time analytical chemistry, size discrimination of neutral polymers, oligosaccharides, oligonucleotides and peptides at monomeric resolution, sequence identification, chemical modification on DNA, potential biomarkers detection and protein folding analysis. This review focuses, on the results obtained with aerolysin nanopores on the fields of chemistry, biology, physics and biotechnology. We discuss and compare as well the results obtained with other protein channel sensors.For the past two decades, the number of papers and the citations in the field of nanopores has increased exponentially. Nanopore technology has become a sensitive, selective, low cost, label-free, real-time and transportable tool for sensing a wide variety of molecules, including ions, polymers, polyelectrolytes, viruses, ligand-molecule complexes and biomolecules. It allows for the analysis of transport properties, conformations, folding, size, sequence or chemical modifications 1-6 . At first, the protein channels were the main sensors used to perform numerous studies 7-9 . Thanks to material science, chemistry, nanoscience and molecular biology, it is now possible to design and manufacture new classes of nanopores: solid-state sensors 10,11 , DNA origami nanopores 12,13 , carbon 14,15 or cyclodextrin nanotubes [16][17][18] , hybrid nanopores 19-21 and glass 22,23 or quartz nanopores 24 with DNA aptamers 25 . The increased interest in nanopore research has been mainly associated to the ultra-fast DNA sequencing challenge, recently achieved by Oxford Nanopore Technology [26][27][28][29] . The objectives at the horizon of this field comprise of proteomic sequencing 6,30-33 , biomarker detection (of micro-RNAs 34 as well as infinitesimal peptides and proteins quantities) and single-molecule mass spectrometry 32,[35][36][37][38][39][40][41][42] . Up to now, the best sensitivity for biotechnological or heath applications is obtained through biological channels.A member of the pore-forming toxin (PFTs) 43 family, aerolysin is a beta structure toxin that has recently been at the center of extensive fundamental studies and some biotechnology applications 32,38,39,[44][45][46][47][48] . In this review we first introduce the molecular mechanism of channel formation, from soluble inactive monomers to a functional pore into a lipid membrane (figure 1); we delve into the structure of monomeric and heptameric aerolysin along with...

Lysenin Channels as Sensors for Ions and Molecules

“…The principle of sensing, a direct expansion of the long-revered Coulter measuring method at the nano-scale [65], is deceptively simple: the resistive pulse technique relies on recording the changes of the ionic currents established through a nanopore when single molecules are electrophoretically driven through the conducting pathway. This research field was initiated by using α-hemolysin as a prototype pore; however, scientists developed and utilized a large variety of synthetic and biological nanopores for similar purposes [6,8,14,33,34,[66][67][68][69][70][71][72][73][74][75][76][77][78][79]. The great interest in this topic is fueled by the promise of fast and reliable sequencing of nucleic acids and peptides [14,35,73,74,76,80], development of sensors for single molecule detection and characterization, fast and reliable determination of biomolecules in complex biological samples, and many other analytical applications.…”

“…The ability of pore-forming proteins and peptides to establish conducting pathways between two sides of a lipid membrane was exploited for decades for numerous analytical applications [1][2][3][4][5][6][7][8][9]. The most common sensing principle relies on measuring changes in the ionic currents elicited by specific and non-specific interactions between analytes of interest and wild-type or engineered protein channels [10][11][12][13][14][15][16][17].…”

“…These tiny nano-scale analytical tools present a high electrical gain, hence detection is straightforward with relatively simple amplifiers. Modulation of ionic currents may occur because of selectivity, existence of regulatory mechanisms that lead to conformational changes and conductance adjustments, and diminished ionic flows resulting from analyte binding or translocation through the pore [6,8,15,[18][19][20].…”

“…Numerous nanopores of biological origin were investigated for sensing applications, such as α-hemolysin, aerolysin, E. coli ClyA toxin, lysenin, and motor proteins [5,6,8,[29][30][31][32][33][34][35][36]. Among those biological tools, lysenin channels are attractive candidates for sensor development owing to their commercial availability, facile reconstitution into artificial membranes, extended stability, intrinsic regulatory mechanisms, and a large unobstructed opening.…”

The Promise of Nanopore Technology: Advances in the Discrimination of Protein Sequences and Chemical Modifications

“…[50][51][52][53] To illustrate the principle of data analysis for peptide sequence analysis, we give an example with one protein channel: the aerolysin. [54,55] This method is not specific to this channel and can be used with other nanopore types, including solid state nanopores, [56][57][58][59][60] hybrid nanopores [61] or track-etched nanopores. [62] The Aerolysin channel is characterized by a strong confinement (≈1.2 nm) and a strong internal charge.…”