Abstract:The study of factors essential for protein-peptide interactions and protein pore-mediated peptide transport are of particular relevance in biology. Wild-type α-hemolysin was adopted as a "nanoreactor" in which perturbations of the current through a protein containing a lumen-residing, aryl-capped antimicrobial peptide were seen for the first time and studied at the single-molecule level. Energy and steric considerations hint that Met-aryl interactions between aromatic residues placed at a peptide's extremities… Show more
“…1). As reported before1920, we attribute these spikes to molecular events whereby a single peptide enters the protein β-barrel and induces reversible partial blocks of the ionic current through steric occlusions of the ion permeation pathway, before getting released to the cis side of the membrane under the influence of the potential imposed across the pore, positive on the peptide side addition. We stress that in all the analyses we excluded the distinct population of very fast occurring, low-amplitude spikes, which reflected blockage events due to peptide bumping into the pore mouth, and not the actual capture events of the peptide into the inner region of the α - HL′s β-barrel that eventually lead to the electrophoretic translocation of the peptide to the cis side of the membrane.…”
Section: Resultssupporting
confidence: 59%
“…Single molecule electrophysiology experiments were performed as previously described2065. Planar lipid membranes made of 1,2-diphytanoyl-sn-glycerophosphocholine (Avanti Polar Lipids, Alabaster, AL) were obtained using the Montal–Mueller technique7626 across a 1:10 hexadecane/pentane (HPLC-grade, Sigma–Aldrich, Germany) pretreated, ~120 μm in diameter orifice punctured on a 25 μm-thick Teflon film (Goodfellow, Malvern, MA) that separated the cis (grounded) and trans chambers of the recording cell.…”
Section: Methodsmentioning
confidence: 99%
“…The large majority of the studies focusing on the molecular details of protein and peptide unfolding or translocation through nanopores employed solid-state nanopores as model translocation systems151617 graphene nanopores18 and a variety of protein channels including α - hemolysin (α - HL)19202122232425, OmpF2627, aerolysin2829, the MspA channel30 or the Phi29 connector channel31. The potential of resistive-pulse technique, in effect tracing structural dynamics from the time series recordings of the single-pore current, was originally revealed by sensing single polymers in buffers32, and was soon followed by the proof of the principle demonstration of the possibility to detect RNA and DNA molecules33, and to separate molecules at high resolution343536.…”
The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.
“…1). As reported before1920, we attribute these spikes to molecular events whereby a single peptide enters the protein β-barrel and induces reversible partial blocks of the ionic current through steric occlusions of the ion permeation pathway, before getting released to the cis side of the membrane under the influence of the potential imposed across the pore, positive on the peptide side addition. We stress that in all the analyses we excluded the distinct population of very fast occurring, low-amplitude spikes, which reflected blockage events due to peptide bumping into the pore mouth, and not the actual capture events of the peptide into the inner region of the α - HL′s β-barrel that eventually lead to the electrophoretic translocation of the peptide to the cis side of the membrane.…”
Section: Resultssupporting
confidence: 59%
“…Single molecule electrophysiology experiments were performed as previously described2065. Planar lipid membranes made of 1,2-diphytanoyl-sn-glycerophosphocholine (Avanti Polar Lipids, Alabaster, AL) were obtained using the Montal–Mueller technique7626 across a 1:10 hexadecane/pentane (HPLC-grade, Sigma–Aldrich, Germany) pretreated, ~120 μm in diameter orifice punctured on a 25 μm-thick Teflon film (Goodfellow, Malvern, MA) that separated the cis (grounded) and trans chambers of the recording cell.…”
Section: Methodsmentioning
confidence: 99%
“…The large majority of the studies focusing on the molecular details of protein and peptide unfolding or translocation through nanopores employed solid-state nanopores as model translocation systems151617 graphene nanopores18 and a variety of protein channels including α - hemolysin (α - HL)19202122232425, OmpF2627, aerolysin2829, the MspA channel30 or the Phi29 connector channel31. The potential of resistive-pulse technique, in effect tracing structural dynamics from the time series recordings of the single-pore current, was originally revealed by sensing single polymers in buffers32, and was soon followed by the proof of the principle demonstration of the possibility to detect RNA and DNA molecules33, and to separate molecules at high resolution343536.…”
The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.
“…2). The time intervals between successive blockade events (τ on ), the blockade extent (ΔI block ) and duration (τ off ) were statistically analysed within the general theory of Markov processes 48, 49 .Figure 2Uni-molecular view of PAMAM-G1 – α-HL interactions at acidic and neutral pH, from electrophysiology traces. Selected ionic current recordings reflecting the PAMAM-G1 – α-HL interactions, and all-events histograms, recorded at ΔV = +100 mV, in 1 M KCl, at acidic (pH = 3; panel a) and neutral pH (pH = 7; panel c).…”
Herein, we describe at uni-molecular level the interactions between poly(amidoamine) (PAMAM) dendrimers of generation 1 and the α-hemolysin protein nanopore, at acidic and neutral pH, and ionic strengths of 0.5 M and 1 M KCl, via single-molecule electrical recordings. The results indicate that kinetics of dendrimer-α-hemolysin reversible interactions is faster at neutral as compared to acidic pH, and we propose as a putative explanation the fine interplay among conformational and rigidity changes on the dendrimer structure, and the ionization state of the dendrimer and the α-hemolysin. From the analysis of the dendrimer’s residence time inside the nanopore, we posit that the pH- and salt-dependent, long-range electrostatic interactions experienced by the dendrimer inside the ion-selective α-hemolysin, induce a non-Stokesian diffusive behavior of the analyte inside the nanopore. We also show that the ability of dendrimer molecules to adapt their structure to nanoscopic spaces, and control the flow of matter through the α-hemolysin nanopore, depends non-trivially on the pH- and salt-induced conformational changes of the dendrimer.
“…Such jamming analyses have been applied to study protein folding in aqueous solution (Jose & Andricioaei, 2012). Accelerated MD simulations captured the atomistic details of these jammed states of a single unstructured peptide molecule, CAMA (Asandei et al, 2011; Mereuta et al, 2014; Mereuta et al, 2012), as it traverses the pore during a translocation event, jamming signatures of which were further compared with that of the same peptide when in free state without the physical constriction of the pore.…”
Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes, receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function and dynamics of membrane proteins.
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