scholarly journals Decoding assembly of alpha-helical transmembrane pores through intermediate states

2021 ◽  
Author(s):  
Neethu Puthumadathil ◽  
Greeshma S Nair ◽  
Smrithi R Krishnan ◽  
Kozhinjampara R Mahendran
Keyword(s):  
Pore Formation ◽  
Single Channel ◽  
Sequence Specificity ◽  
Assembly Mechanism ◽  
Intermediate States ◽  
Transmembrane Pores ◽  
Voltage Dependent ◽  
Structural Assembly ◽  
Dynamics Simulations ◽  
Pore Forming Proteins

AbstractMembrane-active pore-forming alpha-helical peptides and proteins are well known for their dynamic assembly mechanism and it has been critical to delineate the pore-forming structures in the membrane. Previously, attempts have been made to elucidate their assembly mechanism and there is a large gap due to complex pathways by which these membrane-active pores impart their effect. Here we demonstrate the multi-step structural assembly pathway of alpha-helical peptide pores formed by a 37 amino-acid synthetic peptide, pPorU based on the natural porin from Corynebacterium urealyticum using single-channel electrical recordings. More specifically, we report detectable intermediates states during membrane insertion and pore formation of pPorU. The fully assembled pore is functional and exhibited unusually large stable conductance and voltage-dependent gating, generally applicable to a range of pore-forming proteins. Furthermore, we used rationally designed mutants to understand the role of specific amino acids in the assembly of these peptide pores. Mutant peptides that differ from wild-type peptides produced noisy, unstable intermediate states and low conductance pores, demonstrating sequence specificity in the pore-formation process supported by molecular dynamics simulations. We suggest that our study contributes to understanding the mechanism of action of alpha-helical pores and antimicrobial peptides and should be of broad interest to bioengineers to build peptide-based nanopore sensors.

scholarly journals Molecular mechanism of pore formation by aerolysin-like proteins

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160209 ◽  
Author(s):  
Marjetka Podobnik ◽  
Matic Kisovec ◽  
Gregor Anderluh
Keyword(s):  
Cell Biology ◽  
Pore Formation ◽  
Target Cells ◽  
Membrane Pores ◽  
Surface Molecules ◽  
Structural Details ◽  
Transmembrane Pores ◽  
Future Drug ◽  
Pore Forming Proteins ◽  
Unique Domain

Aerolysin-like pore-forming proteins are an important family of proteins able to efficiently damage membranes of target cells by forming transmembrane pores. They are characterized by a unique domain organization and mechanism of action that involves extensive conformational rearrangements. Although structures of soluble forms of many different members of this family are well understood, the structures of pores and their mechanism of assembly have been described only recently. The pores are characterized by well-defined β-barrels, which are devoid of any vestibular regions commonly found in other protein pores. Many members of this family are bacterial toxins; therefore, structural details of their transmembrane pores, as well as the mechanism of pore formation, are an important base for future drug design. Stability of pores and other properties, such as specificity for some cell surface molecules, make this family of proteins a useful set of molecular tools for molecular recognition and sensing in cell biology. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

Sequence Specificity Despite Intrinsic Disorder: How a Disease-Associated Val/Met Polymorphism Shifts Tertiary Interactions in a Long Disordered Protein

2019 ◽  
Author(s):  
Ruchi Lohia ◽  
Reza Salari ◽  
Grace Brannigan
Keyword(s):  
Secondary Structure ◽  
Electrostatic Interactions ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Sequence Specificity ◽  
Intrinsically Disordered ◽  
Specific Effects ◽  
Heterogeneous Polymer ◽  
Non Local ◽  
Dynamics Simulations

<div>The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) encodes a hydrophobic-to-hydrophobic mutation at the midpoint of the prodomain of precursor brain-derived neurotrophic factor (BDNF), one of the earliest SNPs to be associated with neuropsychiatric disorders, for which the underlying molecular mechanism is unknown. Here we report on over 250 μs of fully-atomistic, explicit solvent, temperature replica exchange molecular dynamics simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence.</div><div>The simulations were able to correctly reproduce the location of both local and non-local secondary changes due to the Val66Met mutation when compared with NMR spectroscopy. We find that the local structure change is mediated via entropic and sequence specific effects. We show that the highly disordered prodomain can be meaningfully divided into domains based on sequence alone. Monte Carlo simulations of a self-excluding heterogeneous polymer, with monomers representing each domain, suggest the sequence would be effectively segmented by the long, highly disordered polyampholyte near the sequence midpoint. This is qualitatively consistent with observed interdomain contacts within the BDNF prodomain, although contacts between the two segments are enriched relative to the self-excluding polymer. The Val66Met mutation increases interactions across the boundary between the two segments, due in part to a specific Met-Met interaction with a Methionine in the other segment. This effect propagates to cause the non-local change in secondary structure around the second methionine, previously observed in NMR. The effect is not mediated simply via changes in inter-domain contacts but is also dependent on secondary structure formation around residue 66, indicating a mechanism for secondary structure coupling in disordered proteins. </div>

scholarly journals Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells

1998 ◽  
Vol 111 (2) ◽  
pp. 363-379 ◽  
Author(s):  
Izumi Sugihara
Keyword(s):  
Dissociation Constant ◽  
Time Constant ◽  
Hair Cells ◽  
Single Channel ◽  
Hill Coefficient ◽  
K Channels ◽  
Voltage Dependent ◽  
Time Courses ◽  
The Hill ◽  
Inside Out

Effects of internal Sr2+ on the activity of large-conductance Ca2+-activated K+ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr2+ was approximately one-fourth as potent as Ca2+ in activating these channels. Although the Hill coefficient for Sr2+ was smaller than that for Ca2+, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca2+ and Sr2+. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr2+ at higher concentrations (&gt;100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr2+ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τl−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τb). A significant decrease in τb and no large changes in τl−s were observed with increased Sr2+ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr2+ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba2+ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3).

scholarly journals The Na+/Ca2+ Exchanger Inhibitor, KB-R7943, Blocks a Nonselective Cation Channel Implicated in Chemosensory Transduction

2009 ◽  
Vol 101 (3) ◽  
pp. 1151-1159 ◽  
Author(s):  
A. Pezier ◽  
Y. V. Bobkov ◽  
B. W. Ache
Keyword(s):  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
Trpc Channels ◽  
Dependent Manner ◽  
Open Probability ◽  
Olfactory Transduction ◽  
Voltage Dependent ◽  
Chemosensory Transduction ◽  
Transient Receptor

The mechanism(s) of olfactory transduction in invertebrates remains to be fully understood. In lobster olfactory receptor neurons (ORNs), a nonselective sodium-gated cation (SGC) channel, a presumptive transient receptor potential (TRP)C channel homolog, plays a crucial role in olfactory transduction, at least in part by amplifying the primary transduction current. To better determine the functional role of the channel, it is important to selectively block the channel independently of other elements of the transduction cascade, causing us to search for specific pharmacological blockers of the SGC channel. Given evidence that the Na+/Ca2+ exchange inhibitor, KB-R7943, blocks mammalian TRPC channels, we studied this probe as a potential blocker of the lobster SGC channel. KB-R7943 reversibly blocked the SGC current in both inside- and outside-out patch recordings in a dose- and voltage-dependent manner. KB-R7943 decreased the channel open probability without changing single channel amplitude. KB-R7943 also reversibly and in a dose-dependent manner inhibited both the odorant-evoked discharge of lobster ORNs and the odorant-evoked whole cell current. Our findings strongly imply that KB-R7943 potently blocks the lobster SGC channel and likely does so directly and not through its ability to block the Na+/Ca2+ exchanger.

Single calcium channels in resistance-sized cerebral arteries from rats

1993 ◽  
Vol 264 (2) ◽  
pp. H470-H478 ◽  
Author(s):  
J. M. Quayle ◽  
J. G. McCarron ◽  
J. R. Asbury ◽  
M. T. Nelson
Keyword(s):  
Steady State ◽  
Calcium Channels ◽  
Single Channel ◽  
Cerebral Arteries ◽  
Membrane Depolarization ◽  
Barium Concentration ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Wistar Kyoto ◽  
Single Calcium Channels

Unitary currents through single calcium channels were measured from cell-attached patches on smooth muscle cells isolated from resistance-sized branches of posterior cerebral arteries from Wistar-Kyoto normotensive rats. Barium (80 and 10 mM) was used as the charge carrier, with and without the dihydropyridine calcium channel agonist BAY R 5417. Unitary currents decreased on membrane depolarization, with a slope conductance of 19.4 pS (80 mM barium). Channel open-state probability (Po) was steeply voltage dependent. Peak Po during test pulses from -70 mV increased e-fold per 4.5-mV depolarization. Mean peak Po at potentials positive to +10 mV was 0.44. Po at steady membrane potentials was also steeply voltage dependent, changing e-fold per 4.5 mV in the absence of inactivation. Steady-state Po at positive potentials was substantially lower than peak Po elicited by test pulses, suggesting that steady-state inactivation can reduce Po by as much as 10-fold. Membrane depolarization decreased the longest mean closed time but had little effect on the mean open time of single calcium channels measured during steady-state recordings. Lowering the external barium concentration from 80 to 10 mM reduced the single channel conductance to 12.4 pS and shifted the relationship between steady-state Po and membrane potential by about -30 mV. BAY R 5417 also shifted this relationship by about -15 mV.

scholarly journals Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes

1998 ◽  
Vol 275 (3) ◽  
pp. C646-C652 ◽  
Author(s):  
Guy Droogmans ◽  
Jean Prenen ◽  
Jan Eggermont ◽  
Thomas Voets ◽  
Bernd Nilius
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Anion Channel ◽  
Anion Channels ◽  
Channel Conductance ◽  
Open Channel Block ◽  
Maximum Inhibition ◽  
Voltage Dependent ◽  
A Site ◽  
Maximal Block

We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 ± 2.1 pS ( n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 μM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.

Genetically-Engineered Protease-Activated Triggers in a Pore-Forming Protein

1993 ◽  
Vol 330 ◽  
Author(s):  
Barbara Walker ◽  
Nathan Walsh ◽  
Hagan Bayley
Keyword(s):  
Genetic Engineering ◽  
Cancer Cells ◽  
Pore Formation ◽  
Polypeptide Chain ◽  
Genetically Engineered ◽  
Recognition Sequence ◽  
Selective Activation ◽  
Selective Destruction ◽  
Pore Forming Proteins ◽  
Central Loop

ABSTRACTProtease-activated triggers have been introduced Into a pore-forming protein, staphylococcal a-hemolysin (αHL). The hemolysin was remodeled by genetic engineering to form two-chain constructs with redundant polypeptide sequences at the central loop, the Integrity of which Is crucial for efficient pore formation. The new hemolysins are activated when the polypeptide extensions are removed by proteases. By alterating the protease recognition sequence in the loop, selective activation by specified proteases can be obtained. Protease-triggered pore-forming proteins might be used for the selective destruction of cancer cells that bear tumor-associated proteases. When certain two-chain constructs are treated with proteases, a full-length polypeptide chain forms as the result of a protease-mediated transpeptidation reaction. This reaction might be used to produce chimeric hemolysins that are Inaccessible by conventional routes.

scholarly journals Dynamic Interaction of Norfloxacin to Magnesium Monitored by Bacterial Outer Membrane Nanopores

2020 ◽  
Author(s):  
Jiajun Wang ◽  
Jigneshkumar Dahyabhai Prajapati ◽  
Ulrich Kleinekathöfer ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Bilayers ◽  
Brownian Dynamics ◽  
Single Channel ◽  
Ion Selectivity ◽  
Computational Modelling ◽  
Free Energy Calculations ◽  
Divalent Ions ◽  
Dynamics Simulations ◽  
Brownian Dynamics Simulations

The effect of divalent ions on the permeability of norfloxacin across the major outer membrane channels from <i>E. coli</i> (OmpF, OmpC) and <i>E. aerogenes</i> (Omp35, Omp36) has been investigated at the single channel level. To understand the rate limiting steps in permeation, we reconstituted single porin into planar lipid bilayers and analyzed the ion current fluctuations caused in the presence of norfloxacin. To obtain an atomistic view, we complemented the experiments with millisecond-long free energy calculations based on temperature-accelerated Brownian dynamics simulations to identify the most probable permeation pathways of the antibiotics through the respective pore. Both, experimental analysis and computational modelling, suggest that norfloxacin is able to permeate through the larger porins, i.e., OmpF, OmpC, and Omp35, whereas it only binds to the slightly narrower porin Omp36. Moreover, divalent ions can bind to negatively charged residues inside the porin, reversing the ion selectivity of the pore. In addition, the divalent ions can chelate with the fluoroquinolones and alter their physicochemical properties. The results suggest that the conjugation must break with either one of them when the antibiotics molecules bypass the lumen of the porin, with the conjugation to the antibiotic being more stable than that to the pore. In general, the permeation or binding process of fluoroquinolone in porins occurs irrespective of the presence of divalent ions, but the presences of divalent ions can vary the kinetics significantly.

scholarly journals Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor

2020 ◽  
Vol 152 (9) ◽  
Author(s):  
Kathiresan Natarajan ◽  
Nuriya Mukhtasimova ◽  
Jeremías Corradi ◽  
Matías Lasala ◽  
Cecilia Bouzat ◽  
...  
Keyword(s):  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Single Channel ◽  
Structural Motif ◽  
Site Mutation ◽  
Α7 Nicotinic Acetylcholine Receptor ◽  
Nicotinic Acetylcholine ◽  
Low Concentrations ◽  
Channel Opening ◽  
Dynamics Simulations

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.

Sign in / Sign up

Export Citation Format

Share Document