119 results on '"Paul Blount"'
Search Results
2. The Effects of Airflow on the Mechanosensitive Channels of Escherichia coli MG1655 and the Impact of Survival Mechanisms Triggered
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Violette I. Ramirez, Robin Wray, Paul Blount, and Maria D. King
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airflow stressors ,AMR genes ,bioaerosol resuspension ,mechanosensitive channels ,Biology (General) ,QH301-705.5 - Abstract
Understanding how bacteria respond to ventilated environments is a crucial concept, especially when considering accurate airflow modeling and detection limits. To properly design facilities for aseptic conditions, we must minimize the parameters for pathogenic bacteria to thrive. Identifying how pathogenic bacteria continue to survive, particularly due to their multi-drug resistance characteristics, is necessary for designing sterile environments and minimizing pathogen exposure. A conserved characteristic among bacterial organisms is their ability to maintain intracellular homeostasis for survival and growth in hostile environments. Mechanosensitive (MS) channels are one of the characteristics that guide this phenomenon. Interestingly, during extreme stress, bacteria will forgo favorable homeostasis to execute fast-acting survival strategies. Physiological sensors, such as MS channels, that trigger this survival mechanism are not clearly understood, leaving a gap in how bacteria translate physical stress to an intracellular response. In this paper, we study the role of mechanosensitive ion channels that are potentially triggered by aerosolization. We hypothesize that change in antimicrobial uptake is affected by aerosolization stress. Bacteria regulate their defense mechanisms against antimicrobials, which leads to varying susceptibility. Based on this information we hypothesize that aerosolization stress affects the antimicrobial resistance defense mechanisms of Escherichia coli (E. coli). We analyzed the culturability of knockout E. coli strains with different numbers of mechanosensitive channels and compared antibiotic susceptibility under stressed and unstressed airflow conditions. As a result of this study, we can identify how the defensive mechanisms of resistant bacteria are triggered for their survival in built environments. By changing ventilation airflow velocity and observing the change in antibiotic responses, we show how pathogenic bacteria respond to ventilated environments via mechanosensitive ion channels.
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- 2023
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3. Curcumin activation of a bacterial mechanosensitive channel underlies its membrane permeability and adjuvant properties.
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Robin Wray, Irene Iscla, and Paul Blount
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Curcumin, a natural compound isolated from the rhizome of turmeric, has been shown to have antibacterial properties. It has several physiological effects on bacteria including an apoptosis-like response involving RecA, membrane permeabilization, inhibiting septation, and it can also work synergistically with other antibiotics. The mechanism by which curcumin permeabilizes the bacterial membrane has been unclear. Most bacterial species contain a Mechanosensitive channel of large conductance, MscL, which serves the function of a biological emergency release valve; these large-pore channels open in response to membrane tension from osmotic shifts and, to avoid cell lysis, allow the release of solutes from the cytoplasm. Here we show that the MscL channel underlies the membrane permeabilization by curcumin as well as its synergistic properties with other antibiotics, by allowing access of antibiotics to the cytoplasm; MscL also appears to have an inhibitory role in septation, which is enhanced when activated by curcumin.
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- 2021
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4. Activation of a Bacterial Mechanosensitive Channel, MscL, Underlies the Membrane Permeabilization of Dual-Targeting Antibacterial Compounds
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Robin Wray, Junmei Wang, Paul Blount, and Irene Iscla
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bacterial channels ,antibiotic resistance ,bacterial drug target ,dual mechanism antibiotics ,druggable target ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Resistance to antibiotics is a serious and worsening threat to human health worldwide, and there is an urgent need to develop new antibiotics that can avert it. One possible solution is the development of compounds that possess multiple modes of action, requiring at least two mutations to acquire resistance. Compound SCH-79797 both avoids resistance and has two mechanisms of action: one inhibiting the folate pathway, and a second described as “membrane permeabilization”; however, the mechanism by which membranes from bacterial cells, but not the host, are disrupted has remained mysterious. The opening of the bacterial mechanosensitive channel of large conductance, MscL, which ordinarily serves the physiological role of osmotic emergency release valves gated by hypoosmotic shock, has been previously demonstrated to affect bacterial membrane permeabilization. MscL allows the rapid permeabilization of both ions and solutes through the opening of the largest known gated pore, which has a diameter of 30 Å. We found that SCH-79797 and IRS-16, a more potent derivative, directly bind to the MscL channel and produce membrane permeabilization as a result of its activation. These findings suggest that possessing or adding an MscL-activating component to an antibiotic compound could help to lower toxicity and evade antibiotic resistance.
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- 2022
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5. A native cell membrane nanoparticles system allows for high-quality functional proteoliposome reconstitution
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Limin Yang, Claudio Catalano, Yunyao Xu, Weihua Qiu, Dongyu Zhang, Ann McDermott, Youzhong Guo, and Paul Blount
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KcsA ,MscL ,MscS ,Triggered-release ,NCMN ,Proteoliposome ,Biochemistry ,QD415-436 ,Genetics ,QH426-470 - Abstract
Proteoliposomes mimic the cell membrane environment allowing for structural and functional membrane protein analyses as well as antigen presenting and drug delivery devices. To make proteoliposomes, purified functional membrane proteins are required. Detergents have traditionally been used for the first step in this process. However, they can irreversibly denature or render membrane proteins unstable, and the necessary removal of detergents after reconstitution can decrease proteoliposome yields. The recently developed native cell membrane nanoparticles (NCMN) system has provided a variety of detergent-free alternatives for membrane protein preparation for structural biology research. Here we attempt to employ the MCMN system for the functional reconstitution of channels into proteoliposomes. NCMN polymers NCMNP1-1 and NCMNP7-1, members of a NCMN polymer library that have been successful in extraction and affinity purification of a number of intrinsic membrane proteins, were selected for the purification and subsequent reconstitution of three bacterial channels: KcsA and the mechanosensitive channels of large and small conductance (MscL and MscS). We found that channels in NCMN particles, which appeared to be remarkably stable when stored at 4 °C, can be reconstituted into bilayers by simply incubating with lipids. We show that the resulting proteoliposomes can be patched for electrophysiological studies or used for the generation of liposome-based nanodevices. In sum, the findings demonstrate that the NCMN system is a simple and robust membrane protein extraction and reconstitution approach for making high-quality functional proteoliposomes that could significantly impact membrane protein research and the development of nanodevices.
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- 2021
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6. In Silico Screen Identifies a New Family of Agonists for the Bacterial Mechanosensitive Channel MscL
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Robin Wray, Paul Blount, Junmei Wang, and Irene Iscla
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bacterial channels ,antibiotic resistance ,bacterial drug target ,Therapeutics. Pharmacology ,RM1-950 - Abstract
MscL is a highly conserved mechanosensitive channel found in the majority of bacterial species, including pathogens. It functions as a biological emergency release valve, jettisoning solutes from the cytoplasm upon acute hypoosmotic stress. It opens the largest known gated pore and has been heralded as an antibacterial target. Although there are no known endogenous ligands, small compounds have recently been shown to specifically bind to and open the channel, leading to decreased cell growth and viability. Their binding site is at the cytoplasmic/membrane and subunit interfaces of the protein, which has been recently been proposed to play an essential role in channel gating. Here, we have targeted this pocket using in silico screening, resulting in the discovery of a new family of compounds, distinct from other known MscL-specific agonists. Our findings extended the study of this functional region, the progression of MscL as a viable drug target, and demonstrated the power of in silico screening for identifying and improving the design of MscL agonists.
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- 2022
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7. Novel MscL agonists that allow multiple antibiotics cytoplasmic access activate the channel through a common binding site.
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Robin Wray, Junmei Wang, Irene Iscla, and Paul Blount
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Medicine ,Science - Abstract
The antibiotic resistance crisis is becoming dire, yet in the past several years few potential antibiotics or adjuvants with novel modes of action have been identified. The bacterial mechanosensitive channel of large conductance, MscL, found in the majority of bacterial species, including pathogens, normally functions as an emergency release valve, sensing membrane tension upon low-osmotic stress and discharging cytoplasmic solutes before cell lysis. Opening the huge ~30Å diameter pore of MscL inappropriately is detrimental to the cell, allowing solutes from and even passage of drugs into to cytoplasm. Thus, MscL is a potential novel drug target. However, there are no known natural agonists, and small compounds that modulate MscL activity are just now being identified. Here we describe a small compound, K05, that specifically modulates MscL activity and we compare results with those obtained for the recently characterized MscL agonist 011A. While the structure of K05 only vaguely resembles 011A, many of the findings, including the binding pocket, are similar. On the other hand, both in vivo and molecular dynamic simulations indicate that the two compounds modulate MscL activity in significantly different ways.
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- 2020
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8. Cryo-EM Structure of Mechanosensitive Channel YnaI Using SMA2000: Challenges and Opportunities
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Claudio Catalano, Danya Ben-Hail, Weihua Qiu, Paul Blount, Amedee des Georges, and Youzhong Guo
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YnaI ,SMA2000 ,NCMN ,cryo-EM ,Mechanosensitive Channel ,Chemical technology ,TP1-1185 ,Chemical engineering ,TP155-156 - Abstract
Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, the complete structure of the transmembrane domain of YnaI was not resolved. This reveals a significant limitation of SMA2000 or similar membrane-active copolymers. This limitation may come from the heterogeneity of the polymers and nonspecific interactions between the polymers and the relatively large hydrophobic pockets within the transmembrane domain of YnaI. However, this limitation offers development opportunities for detergent-free technology for challenging membrane proteins.
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- 2021
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9. At What Cost Can Renewable Hydrogen Offset Fossil Fuel Use in Ireland’s Gas Network?
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Tubagus Aryandi Gunawan, Alessandro Singlitico, Paul Blount, James Burchill, James G. Carton, and Rory F. D. Monaghan
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hydrogen ,wind energy ,water electrolysis ,energy storage ,energy system ,geographic information system ,Technology - Abstract
The results of a techno-economic model of distributed wind-hydrogen systems (WHS) located at each existing wind farm on the island of Ireland are presented in this paper. Hydrogen is produced by water electrolysis from wind energy and backed up by grid electricity, compressed before temporarily stored, then transported to the nearest injection location on the natural gas network. The model employs a novel correlation-based approach to select an optimum electrolyser capacity that generates a minimum levelised cost of hydrogen production (LCOH) for each WHS. Three scenarios of electrolyser operation are studied: (1) curtailed wind, (2) available wind, and (3) full capacity operations. Additionally, two sets of input parameters are used: (1) current and (2) future techno-economic parameters. Additionally, two electricity prices are considered: (1) low and (2) high prices. A closest facility algorithm in a geographic information system (GIS) package identifies the shortest routes from each WHS to its nearest injection point. By using current parameters, results show that small wind farms are not suitable to run electrolysers under available wind operation. They must be run at full capacity to achieve sufficiently low LCOH. At full capacity, the future average LCOH is 6–8 €/kg with total hydrogen production capacity of 49 kilotonnes per year, or equivalent to nearly 3% of Irish natural gas consumption. This potential will increase significantly due to the projected expansion of installed wind capacity in Ireland from 5 GW in 2020 to 10 GW in 2030.
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- 2020
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10. Improving the Design of a MscL-Based Triggered Nanovalve
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Paul Blount, Zoltán Kovács, Robin Wray, Juandell Parker, Christina Eaton, and Irene Iscla
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drug-delivery ,nanovalve ,osmoregulation ,biosensor ,hydrophobic gating ,mechanosensor ,Biotechnology ,TP248.13-248.65 - Abstract
The mechanosensitive channel of large conductance, MscL, has been proposed as a triggered nanovalve to be used in drug release and other nanodevices. It is a small homopentameric bacterial protein that has the largest gated pore known: greater than 30 Å. Large molecules, even small proteins can be released through MscL. Although MscL normally gates in response to membrane tension, early studies found that hydrophilic or charged residue substitutions near the constriction of the channel leads to pore opening. Researchers have successfully changed the modality of MscL to open to stimuli such as light by chemically modifying a single residue, G22, within the MscL pore. Here, by utilizing in vivo, liposome efflux, and patch clamp assays we compared modification of G22 with that of another neighboring residue, G26, and demonstrate that modifying G26 may be a better choice for triggered nanovalves used for triggered vesicular release of compounds.
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- 2013
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11. Chimeras Reveal a Single Lipid-Interface Residue that Controls MscL Channel Kinetics as well as Mechanosensitivity
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Li-Min Yang, Dalian Zhong, and Paul Blount
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Biology (General) ,QH301-705.5 - Abstract
MscL, the highly conserved bacterial mechanosensitive channel of large conductance, serves as an osmotic “emergency release valve,” is among the best-studied mechanosensors, and is a paradigm of how a channel senses and responds to membrane tension. Although all homologs tested thus far encode channel activity, many show functional differences. We tested Escherichia coli and Staphylococcus aureus chimeras and found that the periplasmic region of the protein, particularly E. coli I49 and the equivalent S. aureus F47 at the periplasmic lipid-aqueous interface of the first transmembrane domain, drastically influences both the open dwell time and the threshold of channel opening. One mutant shows a severe hysteresis, confirming the importance of this residue in determining the energy barriers for channel gating. We propose that this site acts similarly to a spring for a clasp knife, adjusting the resistance for obtaining and stabilizing an open or closed channel structure.
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- 2013
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12. Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore.
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Robin Wray, Irene Iscla, Ya Gao, Hua Li, Junmei Wang, and Paul Blount
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Biology (General) ,QH301-705.5 - Abstract
The primary mechanism of action of the antibiotic dihydrostreptomycin is binding to and modifying the function of the bacterial ribosome, thus leading to decreased and aberrant translation of proteins; however, the routes by which it enters the bacterial cell are largely unknown. The mechanosensitive channel of large conductance, MscL, is found in the vast majority of bacterial species, where it serves as an emergency release valve rescuing the cell from sudden decreases in external osmolarity. While it is known that MscL expression increases the potency of dihydrostreptomycin, it has remained unclear if this effect is due to a direct interaction. Here, we use a combination of genetic screening, MD simulations, and biochemical and mutational approaches to determine if dihydrostreptomycin directly interacts with MscL. Our data strongly suggest that dihydrostreptomycin binds to a specific site on MscL and modifies its conformation, thus allowing the passage of K+ and glutamate out of, and dihydrostreptomycin into, the cell.
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- 2016
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13. Scanning MscL Channels with Targeted Post-Translational Modifications for Functional Alterations.
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Irene Iscla, Robin Wray, Christina Eaton, and Paul Blount
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Medicine ,Science - Abstract
Mechanosensitive channels are present in all living organisms and are thought to underlie the senses of touch and hearing as well as various important physiological functions like osmoregulation and vasoregulation. The mechanosensitive channel of large conductance (MscL) from Escherichia coli was the first protein shown to encode mechanosensitive channel activity and serves as a paradigm for how a channel senses and responds to mechanical stimuli. MscL plays a role in osmoprotection in E. coli, acting as an emergency release valve that is activated by membrane tension due to cell swelling after an osmotic down-shock. Using an osmotically fragile strain in an osmotic down-shock assay, channel functionality can be directly determined in vivo. In addition, using thiol reagents and expressed MscL proteins with a single cysteine substitution, we have shown that targeted post-translational modifications can be performed, and that any alterations that lead to dysfunctional proteins can be identified by this in vivo assay. Here, we present the results of such a scan performed on 113 MscL cysteine mutants using five different sulfhydryl-reacting probes to confer different charges or hydrophobicity to each site. We assessed which of these targeted modifications affected channel function and the top candidates were further studied using patch clamp to directly determine how channel activity was affected. This comprehensive screen has identified many residues that are critical for channel function as well as highlighted MscL domains and residues that undergo the most drastic environmental changes upon gating.
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- 2015
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14. Mutations in a Conserved Domain of E. coli MscS to the Most Conserved Superfamily Residue Leads to Kinetic Changes.
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Hannah R Malcolm and Paul Blount
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Medicine ,Science - Abstract
In Escherichia coli (E. coli) the mechanosensitive channel of small conductance, MscS, gates in response to membrane tension created from acute external hypoosmotic shock, thus rescuing the bacterium from cell lysis. E. coli MscS is the most well studied member of the MscS superfamily of channels, whose members are found throughout the bacterial and plant kingdoms. Homology to the pore lining helix and upper vestibule domain of E. coli MscS is required for inclusion into the superfamily. Although highly conserved, in the second half of the pore lining helix (TM3B), E. coli MscS has five residues significantly different from other members of the superfamily. In superfamilies such as this, it remains unclear why variations within such a homologous region occur: is it tolerance of alternate residues, or does it define functional variance within the superfamily? Point mutations (S114I/T, L118F, A120S, L123F, F127E/K/T) and patch clamp electrophysiology were used to study the effect of changing these residues in E. coli MscS on sensitivity and gating. The data indicate that variation at these locations do not consistently lead to wildtype channel phenotypes, nor do they define large changes in mechanosensation, but often appear to effect changes in the E. coli MscS channel gating kinetics.
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- 2015
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15. S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: implications for detergent-solubilized membrane proteins.
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Michael R Dorwart, Robin Wray, Chad A Brautigam, Youxing Jiang, and Paul Blount
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Biology (General) ,QH301-705.5 - Abstract
While the bacterial mechanosensitive channel of large conductance (MscL) is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL) is a pentamer, while the S. aureus protein (SaMscL) forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL) is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complementing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering) and established that SaMscL is a pentamer when solubilized in Triton X-100 and C(8)E(5) detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.
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- 2010
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16. Feeling the tension: the bacterial mechanosensitive channel of large conductance as a model system and drug target
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Junmei Wang and Paul Blount
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Physiology ,Physiology (medical) - Published
- 2023
17. An in vivo screen reveals protein‐lipid interactions crucial for gating a mechanosensitive channel
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Irene Iscla, Robin Wray, and Paul Blount
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- 2010
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18. Manipulating the permeation of charged compounds through the MscL nanovalve
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Li‐Min Yang and Paul Blount
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- 2010
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19. Human mutations highlight an intersubunit cation–π bond that stabilizes the closed but not open or inactivated states of TRPV channels
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Jinfeng Teng, Ching Kung, Andriy Anishkin, and Paul Blount
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Protein Conformation, alpha-Helical ,TRPV4 ,Multidisciplinary ,biology ,Protein Stability ,Chemistry ,Mutation, Missense ,Xenopus ,TRPV Cation Channels ,Saccharomyces cerevisiae ,computer.file_format ,Gating ,Protein Data Bank ,biology.organism_classification ,TRPV ,Xenopus laevis ,Transient receptor potential channel ,Amino Acid Substitution ,PNAS Plus ,Biophysics ,Animals ,Humans ,Salt bridge ,computer ,Linker - Abstract
An adequate response of a living cell to the ever-changing environment requires integration of numerous sensory inputs. In many cases, it can be achieved even at the level of a single receptor molecule. Polymodal transient receptor potential (TRP) channels have been shown to integrate mechanical, chemical, electric, and thermal stimuli. Inappropriate gating can lead to pathologies. Among the >60 known TRP vanilloid subfamily (V) 4 mutations that interfere with bone development are Y602C or R616Q at the S4–S5 linker. A cation–π bond between the conservative residues Y602 and R616 of neighboring subunits appears likely in many homologous channel structures in a closed state. Our experiments with TRPV4 mutants indicate that the resting-closed state remains stable while the bond is substituted by a salt bridge or disulfide bond, whereas disruption of the contact by mutations like Y602C or R616Q produces gain-of-function phenotypes when TRPV4 is heterologously expressed in the Xenopus oocyte or yeast. Our data indicate that the Y602–R616 cation–π interactions link the four S4–S5 linker helices together, forming a girdle backing the closed gate. Analogous cation–π bonds and the girdle are seen in many closed TRP channel structures. This girdle is not observed in the cryo-EM structure of amphibian TRPV4 (Protein Data Bank ID code 6BBJ), which appears to be in a different impermeable state—we hypothesize this is the inactivated state.
- Published
- 2019
20. Spectrin couples cell shape, cortical tension, and Hippo signaling in retinal epithelial morphogenesis
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Limin Yang, Duojia Pan, Huiyan Lei, Hua Deng, Pei Wen, and Paul Blount
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macromolecular substances ,Development ,Biology ,Article ,Retina ,Cell membrane ,Adherens junction ,03 medical and health sciences ,0302 clinical medicine ,Cell Signaling ,Cell Adhesion ,Morphogenesis ,medicine ,Animals ,Spectrin ,Cytoskeleton ,Cell Shape ,Actin ,030304 developmental biology ,0303 health sciences ,Epithelial Cells ,Apical constriction ,Cell Biology ,Cell biology ,Drosophila melanogaster ,medicine.anatomical_structure ,Hippo signaling ,030217 neurology & neurosurgery ,Intracellular ,Signal Transduction - Abstract
Deng et al. report an essential role for the spectrin-based membrane skeleton in specifying cell shape by transmitting intracellular actomyosin force to cell membrane. Their findings uncover an essential mechanism that couples cell shape, cortical tension, and Hippo signaling in tissue morphogenesis., Although extracellular force has a profound effect on cell shape, cytoskeleton tension, and cell proliferation through the Hippo signaling effector Yki/YAP/TAZ, how intracellular force regulates these processes remains poorly understood. Here, we report an essential role for spectrin in specifying cell shape by transmitting intracellular actomyosin force to cell membrane. While activation of myosin II in Drosophila melanogaster pupal retina leads to increased cortical tension, apical constriction, and Yki-mediated hyperplasia, spectrin mutant cells, despite showing myosin II activation and Yki-mediated hyperplasia, paradoxically display decreased cortical tension and expanded apical area. Mechanistically, we show that spectrin is required for tethering cortical F-actin to cell membrane domains outside the adherens junctions (AJs). Thus, in the absence of spectrin, the weakened attachment of cortical F-actin to plasma membrane results in a failure to transmit actomyosin force to cell membrane, causing an expansion of apical surfaces. These results uncover an essential mechanism that couples cell shape, cortical tension, and Hippo signaling and highlight the importance of non–AJ membrane domains in dictating cell shape in tissue morphogenesis.
- Published
- 2020
21. Life with Bacterial Mechanosensitive Channels, from Discovery to Physiology to Pharmacological Target
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Irene R Iscla and Paul Blount
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Protein subunit ,Review ,Biology ,Bacterial Physiological Phenomena ,Microbiology ,Bacterial cell structure ,Ion Channels ,03 medical and health sciences ,0302 clinical medicine ,Osmoregulation ,Escherichia coli ,Molecular Biology ,Ion channel ,030304 developmental biology ,0303 health sciences ,Bacteria ,Escherichia coli Proteins ,fungi ,Cell Membrane ,Membrane transport ,Transmembrane protein ,Anti-Bacterial Agents ,Infectious Diseases ,Targeted drug delivery ,Biophysics ,Mechanosensitive channels ,Membrane biophysics ,030217 neurology & neurosurgery - Abstract
SUMMARY General principles in biology have often been elucidated from the study of bacteria. This is true for the bacterial mechanosensitive channel of large conductance, MscL, the channel highlighted in this review. This channel functions as a last-ditch emergency release valve discharging cytoplasmic solutes upon decreases in osmotic environment. Opening the largest gated pore, MscL passes molecules up to 30 A in diameter; exaggerated conformational changes yield advantages for study, including in vivo assays. MscL contains structural/functional themes that recur in higher organisms and help elucidate how other, structurally more complex, channels function. These features of MscL include (i) the ability to directly sense, and respond to, biophysical changes in the membrane, (ii) an α helix (“slide helix”) or series of charges (“knot in a rope”) at the cytoplasmic membrane boundary to guide transmembrane movements, and (iii) important subunit interfaces that, when disrupted, appear to cause the channel to gate inappropriately. MscL may also have medical applications: the modality of the MscL channel can be changed, suggesting its use as a triggered nanovalve in nanodevices, including those for drug targeting. In addition, recent studies have shown that the antibiotic streptomycin opens MscL and uses it as one of the primary paths to the cytoplasm. Moreover, the recent identification and study of novel specific agonist compounds demonstrate that the channel is a valid drug target. Such compounds may serve as novel-acting antibiotics and adjuvants, a way of permeabilizing the bacterial cell membrane and, thus, increasing the potency of commonly used antibiotics.
- Published
- 2020
22. Novel compounds that specifically bind and modulate MscL: insights into channel gating mechanisms
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Robin Wray, Zoltan Kovacs, Paul Blount, Junmei Wang, and Irene R Iscla
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0301 basic medicine ,Osmotic shock ,Protein subunit ,Binding pocket ,Microbial Sensitivity Tests ,Gating ,Molecular Dynamics Simulation ,Mechanotransduction, Cellular ,Biochemistry ,Ion Channels ,03 medical and health sciences ,0302 clinical medicine ,Escherichia coli ,Genetics ,Amino Acid Sequence ,Mode of action ,Molecular Biology ,Sulfonamides ,Binding Sites ,Sequence Homology, Amino Acid ,Channel gating ,Chemistry ,Research ,Escherichia coli Proteins ,fungi ,Anti-Bacterial Agents ,High-Throughput Screening Assays ,Molecular Docking Simulation ,030104 developmental biology ,Cytoplasm ,Mutation ,Biophysics ,Mechanosensitive channels ,Ion Channel Gating ,030217 neurology & neurosurgery ,Biotechnology - Abstract
The bacterial mechanosensitive channel of large conductance (MscL) normally functions as an emergency release valve discharging cytoplasmic solutes upon osmotic stress. Opening the large pore of MscL inappropriately is detrimental to the cell, and thus it has been speculated to be a potential antibiotic target. Although MscL is one of the best studied mechanosensitive channels, no chemical that influenced bacterial growth by modulating MscL is known. We therefore used a high-throughput screen to identify compounds that slowed growth in an MscL-dependent manner. We characterized 2 novel sulfonamide compounds identified in the screen. We demonstrated that, although both increase MscL gating, one of these compounds does not work through the folate pathway, as other antimicrobial sulfonamides; indeed, the sulfonamide portion of the compound is not needed for activity. The only mode of action appears to be MscL activation. The binding pocket is where an α-helix runs along the cytoplasmic membrane and interacts with a neighboring subunit; analogous motifs have been observed in several prokaryotic and eukaryotic channels. The data not only demonstrate that MscL is a viable antibiotic target, but also give insight into the gating mechanisms of MscL, and they may have implications for developing agonists for other channels.-Wray, R., Iscla, I., Kovacs, Z., Wang, J., Blount, P. Novel compounds that specifically bind and modulate MscL: insights into channel gating mechanisms.
- Published
- 2018
23. Curcumin activation of a bacterial mechanosensitive channel underlies its membrane permeability and adjuvant properties
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Irene Iscla, Paul Blount, and Robin Wray
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Cytoplasm ,Cell Membrane Permeability ,Bacillus ,Pathology and Laboratory Medicine ,Biochemistry ,Ion Channels ,Antibiotics ,Medicine and Health Sciences ,Public and Occupational Health ,Membrane Technology ,Biology (General) ,Antimicrobials ,Escherichia coli Proteins ,Drugs ,Neurochemistry ,Neurotransmitters ,Built Structures ,Vaccination and Immunization ,Anti-Bacterial Agents ,Bacterial Pathogens ,Bacillus Subtilis ,Experimental Organism Systems ,Medical Microbiology ,Tetracyclines ,Prokaryotic Models ,Engineering and Technology ,Glutamate ,Cellular Structures and Organelles ,Pathogens ,Research Article ,Curcumin ,Structural Engineering ,QH301-705.5 ,Immunology ,Research and Analysis Methods ,Microbiology ,Membrane Structures ,Microbial Control ,Virology ,Escherichia coli ,Genetics ,Microbial Pathogens ,Molecular Biology ,Pharmacology ,Bacteria ,Antibacterial Therapy ,fungi ,Cell Membrane ,Organisms ,Biology and Life Sciences ,Cell Biology ,RC581-607 ,Animal Studies ,Antibacterials ,Parasitology ,Preventive Medicine ,Immunologic diseases. Allergy ,Neuroscience - Abstract
Curcumin, a natural compound isolated from the rhizome of turmeric, has been shown to have antibacterial properties. It has several physiological effects on bacteria including an apoptosis-like response involving RecA, membrane permeabilization, inhibiting septation, and it can also work synergistically with other antibiotics. The mechanism by which curcumin permeabilizes the bacterial membrane has been unclear. Most bacterial species contain a Mechanosensitive channel of large conductance, MscL, which serves the function of a biological emergency release valve; these large-pore channels open in response to membrane tension from osmotic shifts and, to avoid cell lysis, allow the release of solutes from the cytoplasm. Here we show that the MscL channel underlies the membrane permeabilization by curcumin as well as its synergistic properties with other antibiotics, by allowing access of antibiotics to the cytoplasm; MscL also appears to have an inhibitory role in septation, which is enhanced when activated by curcumin., Author summary The rhizome of turmeric contains a compound, called curcumin, which has been shown to have several biological activities, including antibacterial properties. Previous studies have found that upon curcumin treatment, bacterial cells become “leaky”; their membranes permeabilized. In addition, curcumin appears to work in synergy with several other antibiotics. Finally, it has been observed that curcumin can lead to bacterial cells that can grow but not divide, thus leading to long filamentous cells with multiple copies of their DNA. But the mechanisms underlying these findings were mysterious. Here we show that curcumin targets a specific protein in the bacterial cell envelope that can open a very large pore—the largest regulated pore known in the biological world. This pore usually is only used in extreme situations as an “emergency release valve” that prevents lysis from osmotic stresses. It is the inappropriate opening of this pore by curcumin that leads to the permeation of the bacterial cell envelope, as well as the synergy with other antibiotics by allowing the antibiotics easier access to the inside of the cell, and even the inhibition of bacterial cell division.
- Published
- 2021
24. Interaction of the Mechanosensitive Channel, MscS, with the Membrane Bilayer through Lipid Intercalation into Grooves and Pockets
- Author
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Tim, Rasmussen, Akiko, Rasmussen, Limin, Yang, Corinna, Kaul, Susan, Black, Heloisa, Galbiati, Stuart J, Conway, Samantha, Miller, Paul, Blount, and Ian Rylance, Booth
- Subjects
Models, Molecular ,Biochemical Phenomena ,Cardiolipins ,Protein Conformation ,Escherichia coli Proteins ,Lipid Bilayers ,Tryptophan ,Biological Transport ,Ion Channels ,Article ,Escherichia coli ,lipids (amino acids, peptides, and proteins) ,Protein Interaction Domains and Motifs ,Hydrophobic and Hydrophilic Interactions ,Phospholipids ,Protein Binding - Abstract
All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein-lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even "conservative" hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4.
- Published
- 2019
25. Effects of Low Intensity Focused Ultrasound on Liposomes Containing Channel proteins
- Author
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George N. Saddik, Meghedi Babakhanian, Paul Blount, Warren S. Grundfest, Bryan Nowroozi, Lilian Boodaghians, and Limin Yang
- Subjects
0301 basic medicine ,Mechanotransduction ,1.1 Normal biological development and functioning ,lcsh:Medicine ,Bioengineering ,Neurodegenerative ,Mechanotransduction, Cellular ,Models, Biological ,Article ,Ion Channels ,Cell membrane ,03 medical and health sciences ,0302 clinical medicine ,Models ,Underpinning research ,medicine ,Premovement neuronal activity ,lcsh:Science ,Ion transporter ,Multidisciplinary ,Chemistry ,Vesicle ,lcsh:R ,Cell Membrane ,Neurosciences ,Depolarization ,Biological ,030104 developmental biology ,Membrane ,medicine.anatomical_structure ,Ultrasonic Waves ,Liposomes ,Neurological ,Biophysics ,lcsh:Q ,Mechanosensitive channels ,Cellular ,Ion Channel Gating ,030217 neurology & neurosurgery - Abstract
The ability to reversibly and non-invasively modulate region-specific brain activity in vivo suggests Low Intensity Focused Ultrasound (LIFU) as potential therapeutics for neurological dysfunctions such as epilepsy and Parkinson’s disease. While in vivo studies provide evidence of the bioeffects of LIFU on neuronal activity, they merely hint at potential mechanisms but do not fully explain how this technology achieves these effects. One potential hypothesis is that LIFU produces local membrane depolarization by mechanically perturbing the neuronal cell membrane, or activating channels or other proteins embedded in the membrane. Proteins that sense mechanical perturbations of the membrane, such as those gated by membrane tension, are prime candidates for activating in response to LIFU and thus leading to the neurological responses that have been measured. Here we use the bacterial mechanosensitive channel MscL, which has been purified and reconstituted in liposomes, to determine how LIFU may affect the activation of this membrane-tension gated channel. Two bacterial voltage-gated channels, KvAP and NaK2K F92A channels were also studied. Surprisingly, the results suggest that ultrasound modulation and membrane perturbation does not induce channel gating, but rather induces pore formation at the membrane protein-lipid interface. However, in vesicles with high MscL mechanosensitive channel concentrations, apparent decreases in pore formation are observed, suggesting that this membrane-tension-sensitive protein may serve to increase the elasticity of the membrane, presumably because of expansion of the channel in the plane of the membrane independent of channel gating.
- Published
- 2018
26. At What Cost Can Renewable Hydrogen Offset Fossil Fuel Use in Ireland’s Gas Network?
- Author
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Alessandro Singlitico, Paul Blount, Tubagus Aryandi Gunawan, James Burchill, Rory F.D. Monaghan, and J.G. Carton
- Subjects
Energy storage ,Control and Optimization ,Offset (computer science) ,020209 energy ,Energy Engineering and Power Technology ,02 engineering and technology ,water electrolysis ,lcsh:Technology ,energy system ,hydrogen ,wind energy ,energy storage ,geographic information system ,natural gas network ,Energy system ,Natural gas ,0202 electrical engineering, electronic engineering, information engineering ,SDG 7 - Affordable and Clean Energy ,Electrical and Electronic Engineering ,Wind energy ,Engineering (miscellaneous) ,Hydrogen production ,Wind power ,lcsh:T ,Renewable Energy, Sustainability and the Environment ,business.industry ,Fossil fuel ,Environmental engineering ,Water electrolysis ,021001 nanoscience & nanotechnology ,Natural gas network ,Renewable energy ,Environmental science ,Electricity ,0210 nano-technology ,business ,Geographic information system ,Hydrogen ,Energy (miscellaneous) - Abstract
The results of a techno-economic model of distributed wind-hydrogen systems (WHS) located at each existing wind farm on the island of Ireland are presented in this paper. Hydrogen is produced by water electrolysis from wind energy and backed up by grid electricity, compressed before temporarily stored, then transported to the nearest injection location on the natural gas network. The model employs a novel correlation-based approach to select an optimum electrolyser capacity that generates a minimum levelised cost of hydrogen production (LCOH) for each WHS. Three scenarios of electrolyser operation are studied: (1) curtailed wind, (2) available wind, and (3) full capacity operations. Additionally, two sets of input parameters are used: (1) current and (2) future techno-economic parameters. Additionally, two electricity prices are considered: (1) low and (2) high prices. A closest facility algorithm in a geographic information system (GIS) package identifies the shortest routes from each WHS to its nearest injection point. By using current parameters, results show that small wind farms are not suitable to run electrolysers under available wind operation. They must be run at full capacity to achieve sufficiently low LCOH. At full capacity, the future average LCOH is 6–8 €/kg with total hydrogen production capacity of 49 kilotonnes per year, or equivalent to nearly 3% of Irish natural gas consumption. This potential will increase significantly due to the projected expansion of installed wind capacity in Ireland from 5 GW in 2020 to 10 GW in 2030.
- Published
- 2020
27. Novel MscL agonists that allow multiple antibiotics cytoplasmic access activate the channel through a common binding site
- Author
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Junmei Wang, Irene R Iscla, Paul Blount, and Robin Wray
- Subjects
Staphylococcus ,Cell ,Antibiotics ,Molecular Dynamics ,Pathology and Laboratory Medicine ,Biochemistry ,01 natural sciences ,Ion Channels ,Cytosol ,Computational Chemistry ,Protein structure ,Medicine and Health Sciences ,Biochemical Simulations ,Staphylococcus Aureus ,Amino Acids ,Mycobacteriaceae ,Free Energy ,0303 health sciences ,Multidisciplinary ,010304 chemical physics ,Antimicrobials ,Organic Compounds ,Chemistry ,Escherichia coli Proteins ,Physics ,Drugs ,Neurochemistry ,Neurotransmitters ,Anti-Bacterial Agents ,Bacterial Pathogens ,medicine.anatomical_structure ,Medical Microbiology ,Physical Sciences ,Medicine ,Thermodynamics ,Mechanosensitive channels ,Pathogens ,Glutamate ,Research Article ,Agonist ,medicine.drug_class ,Science ,Glutamic Acid ,Molecular Dynamics Simulation ,Microbiology ,03 medical and health sciences ,In vivo ,Microbial Control ,0103 physical sciences ,Escherichia coli ,medicine ,Sulfur Containing Amino Acids ,Cysteine ,Binding site ,Microbial Pathogens ,030304 developmental biology ,Pharmacology ,Binding Sites ,Bacteria ,Organic Chemistry ,fungi ,Organisms ,Chemical Compounds ,Biology and Life Sciences ,Computational Biology ,Proteins ,Protein Structure, Tertiary ,Cytoplasm ,Antibiotic Resistance ,Potassium ,Biophysics ,Antimicrobial Resistance ,Neuroscience - Abstract
The antibiotic resistance crisis is becoming dire, yet in the past several years few potential antibiotics or adjuvants with novel modes of action have been identified. The bacterial mechanosensitive channel of large conductance, MscL, found in the majority of bacterial species, including pathogens, normally functions as an emergency release valve, sensing membrane tension upon low-osmotic stress and discharging cytoplasmic solutes before cell lysis. Opening the huge ~30Å diameter pore of MscL inappropriately is detrimental to the cell, allowing solutes from and even passage of drugs into to cytoplasm. Thus, MscL is a potential novel drug target. However, there are no known natural agonists, and small compounds that modulate MscL activity are just now being identified. Here we describe a small compound, K05, that specifically modulates MscL activity and we compare results with those obtained for the recently characterized MscL agonist 011A. While the structure of K05 only vaguely resembles 011A, many of the findings, including the binding pocket, are similar. On the other hand, both in vivo and molecular dynamic simulations indicate that the two compounds modulate MscL activity in significantly different ways.
- Published
- 2020
28. A new antibiotic with potent activity targets MscL
- Author
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Uwe H. Stroeher, Edwin S. Tjandra, Colin L. Raston, Nick van Holst, Frederick M. Ausubel, Soumya Ramu, Johnny X. Huang, Annie L. Conery, Mark A. T. Blaskovich, Christopher T. Gibson, Ashley D. Slattery, Ian M. Orme, Mark E. Cooper, Andrés Obregón-Henao, Melissa H. Brown, Irene R Iscla, Jonah Larkins-Ford, Paul Blount, Robin Wray, Angela M. Kavanagh, Cindy Macardle, Ramiz A. Boulos, and Chee Ling Tong
- Subjects
Methicillin-Resistant Staphylococcus aureus ,medicine.drug_class ,Antiparasitic ,In silico ,Antibiotics ,Microbial Sensitivity Tests ,Biology ,Pharmacology ,medicine.disease_cause ,Staphylococcal infections ,Mechanotransduction, Cellular ,Ion Channels ,Microbiology ,Cell Line ,Drug Discovery ,medicine ,Animals ,Humans ,Enzyme Inhibitors ,Caenorhabditis elegans ,Staphylococcal Infections ,Antimicrobial ,biology.organism_classification ,medicine.disease ,Methicillin-resistant Staphylococcus aureus ,3. Good health ,Anti-Bacterial Agents ,Disease Models, Animal ,Treatment Outcome ,Staphylococcus aureus ,Original Article ,Bacteria - Abstract
The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections.
- Published
- 2015
29. On the measurement of pulse recovery times in Gallium Nitride low noise amplifiers
- Author
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Nicholas Novaris, Charles Trantanella, and Paul Blount
- Subjects
010302 applied physics ,Microwave amplifiers ,Materials science ,business.industry ,Amplifier ,020206 networking & telecommunications ,Jamming ,Gallium nitride ,02 engineering and technology ,01 natural sciences ,Low noise ,Pulse (physics) ,chemistry.chemical_compound ,chemistry ,0103 physical sciences ,0202 electrical engineering, electronic engineering, information engineering ,Optoelectronics ,Oscilloscope ,business ,Energy (signal processing) - Abstract
In this paper, we discuss the phenomenon of pulse recovery in overdriven low noise amplifiers fabricated with Gallium Nitride technology. We present our experimental set up to determine the pulse recovery time of a commercially available amplifier, and then present our measured results. We note the recovery time follows a deterministic relationship with input energy that can be described by a radical function.
- Published
- 2017
30. Electrostatics at the membrane define MscL channel mechanosensitivity and kinetics
- Author
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Paul Blount and Dalian Zhong
- Subjects
Osmotic shock ,Molecular Sequence Data ,Static Electricity ,Biology ,Biochemistry ,Ion Channels ,Research Communications ,Cell membrane ,Static electricity ,Genetics ,medicine ,Amino Acid Sequence ,Molecular Biology ,Ion channel ,Sequence Homology, Amino Acid ,Mechanosensation ,Escherichia coli Proteins ,Cell Membrane ,fungi ,Electrostatics ,Kinetics ,Membrane ,medicine.anatomical_structure ,Mutagenesis, Site-Directed ,Biophysics ,Mechanosensitive channels ,Ion Channel Gating ,Biotechnology - Abstract
The bacterial mechanosensitive channel of large conductance (MscL) serves as a biological emergency release valve, preventing the occurrence of cell lysis caused by acute osmotic stress. Its tractable nature allows it to serve as a paradigm for how a protein can directly sense membrane tension. Although much is known of the importance of the hydrophobicity of specific residues in channel gating, it has remained unclear whether electrostatics at the membrane plays any role. We studied MscL chimeras derived from functionally distinct orthologues: Escherichia coli and Staphylococcus aureus. Dissection of one set led to an observation that changing the charge of a single residue, K101, of E. coli (Ec)-MscL, effects a channel phenotype: when mutated to a negative residue, the channel is less mechanosensitive and has longer open dwell times. Assuming electrostatic interactions, we determined whether they are due to protein–protein or protein–lipid interactions by performing site-directed mutagenesis elsewhere in the protein and reconstituting channels into defined lipids, with and without negative head groups. We found that although both interactions appear to play some role, the primary determinant of the channel phenotype seems to be protein–lipid electrostatics. The data suggest a model for the role of electrostatic interactions in the dynamics of MscL gating.—Zhong, D., Blount, P. Electrostatics at the membrane define MscL channel mechanosensitivity and kinetics.
- Published
- 2014
31. Improving the Design of a MscL-Based Triggered Nanovalve
- Author
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Robin Wray, Zoltan Kovacs, Paul Blount, Irene R Iscla, Christina Eaton, and Juandell Parker
- Subjects
Liposome ,nanovalve ,Chemistry ,lcsh:Biotechnology ,Clinical Biochemistry ,drug-delivery ,osmoregulation ,biosensor ,hydrophobic gating ,mechanosensor ,fungi ,Nanotechnology ,General Medicine ,Membrane tension ,Article ,Bacterial protein ,lcsh:TP248.13-248.65 ,Drug delivery ,Drug release ,Biophysics ,Mechanosensitive channels ,Patch clamp - Abstract
The mechanosensitive channel of large conductance, MscL, has been proposed as a triggered nanovalve to be used in drug release and other nanodevices. It is a small homopentameric bacterial protein that has the largest gated pore known: greater than 30 A. Large molecules, even small proteins can be released through MscL. Although MscL normally gates in response to membrane tension, early studies found that hydrophilic or charged residue substitutions near the constriction of the channel leads to pore opening. Researchers have successfully changed the modality of MscL to open to stimuli such as light by chemically modifying a single residue, G22, within the MscL pore. Here, by utilizing in vivo, liposome efflux, and patch clamp assays we compared modification of G22 with that of another neighboring residue, G26, and demonstrate that modifying G26 may be a better choice for triggered nanovalves used for triggered vesicular release of compounds.
- Published
- 2013
32. A High Efficiency, Ka-Band Pulsed Gallium Nitride Power Amplifier for Radar Applications
- Author
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Steven E. Huettner, Paul Blount, and Ben Cannon
- Subjects
010302 applied physics ,Power-added efficiency ,Materials science ,business.industry ,Amplifier ,RF power amplifier ,020206 networking & telecommunications ,Gallium nitride ,02 engineering and technology ,High-electron-mobility transistor ,01 natural sciences ,chemistry.chemical_compound ,Electricity generation ,chemistry ,0103 physical sciences ,0202 electrical engineering, electronic engineering, information engineering ,Optoelectronics ,Ka band ,business ,Monolithic microwave integrated circuit - Abstract
The design and performance of a three stage Ka-band power amplifier MMIC utilizing a 0.2 um GaN on SiC HEMT process technology is presented. Measured both pulsed and CW, the design demonstrates over 5 W of saturated power with an associated power added efficiency (PAE) of 41%. The die size is 2.62x1.62 mm.
- Published
- 2016
33. Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore
- Author
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Hua Li, Junmei Wang, Robin Wray, Paul Blount, Irene R Iscla, and Ya Gao
- Subjects
0301 basic medicine ,Protein Conformation ,Plasma protein binding ,Biochemistry ,Ion Channels ,Cell membrane ,chemistry.chemical_compound ,0302 clinical medicine ,Protein structure ,Antibiotics ,Biochemical Simulations ,Medicine and Health Sciences ,Membrane Technology ,Biology (General) ,Amino Acids ,Crystallography ,Organic Compounds ,Antimicrobials ,General Neuroscience ,Physics ,Escherichia coli Proteins ,Drugs ,Translation (biology) ,Neurochemistry ,Neurotransmitters ,Condensed Matter Physics ,Built Structures ,Lipids ,3. Good health ,Anti-Bacterial Agents ,Molecular Docking Simulation ,Chemistry ,medicine.anatomical_structure ,Physical Sciences ,Crystal Structure ,Engineering and Technology ,Mechanosensitive channels ,Glutamate ,General Agricultural and Biological Sciences ,Dihydrostreptomycin Sulfate ,Ion Channel Gating ,Mechanoreceptors ,Research Article ,Protein Binding ,Structural Engineering ,QH301-705.5 ,Glutamic Acid ,Library Screening ,Biology ,Molecular Dynamics Simulation ,Research and Analysis Methods ,Microbiology ,General Biochemistry, Genetics and Molecular Biology ,Membrane Structures ,03 medical and health sciences ,Microbial Control ,medicine ,Escherichia coli ,Solid State Physics ,Sulfur Containing Amino Acids ,Cysteine ,Molecular Biology Techniques ,Molecular Biology ,Ion channel ,Dihydrostreptomycin ,Pharmacology ,Molecular Biology Assays and Analysis Techniques ,Binding Sites ,General Immunology and Microbiology ,fungi ,Organic Chemistry ,Cell Membrane ,Chemical Compounds ,Biology and Life Sciences ,Computational Biology ,Proteins ,030104 developmental biology ,chemistry ,Mutation ,Biophysics ,Potassium ,030217 neurology & neurosurgery ,Neuroscience - Abstract
The primary mechanism of action of the antibiotic dihydrostreptomycin is binding to and modifying the function of the bacterial ribosome, thus leading to decreased and aberrant translation of proteins; however, the routes by which it enters the bacterial cell are largely unknown. The mechanosensitive channel of large conductance, MscL, is found in the vast majority of bacterial species, where it serves as an emergency release valve rescuing the cell from sudden decreases in external osmolarity. While it is known that MscL expression increases the potency of dihydrostreptomycin, it has remained unclear if this effect is due to a direct interaction. Here, we use a combination of genetic screening, MD simulations, and biochemical and mutational approaches to determine if dihydrostreptomycin directly interacts with MscL. Our data strongly suggest that dihydrostreptomycin binds to a specific site on MscL and modifies its conformation, thus allowing the passage of K+ and glutamate out of, and dihydrostreptomycin into, the cell., The antibiotic dihydrostreptomycin binds to a specific site on the bacterial mechanosensitive channel MscL, opens the pore, and appears to pass through to access the cytoplasm of the cell., Author Summary Streptomycin is one of the original and best studied antibiotics. Its primary mechanism of action is the interference with protein synthesis by binding to and modifying the function of the bacterial ribosome. However, the antibiotic is quite large, bulky, and is charged, so the mechanisms by which it accesses the inside of the bacterial cell have remained largely unknown. Previously, we have found that the expression of a bacterial mechanosensitive channel, MscL, increases the potency of a variant of this antibiotic, dihydrostreptomycin. Here, we define the dihydrostreptomycin binding site on the MscL channel. We also show how this antibiotic modifies the channel and opens the pore, allowing the diffusion of solutes, such as potassium and glutamate, from the cytoplasm of the cell out to the medium. Finally, we provide evidence that dihydrostreptomycin can pass through MscL and that the channel is thus one pathway by which the drug accesses the inside of the bacterial cell.
- Published
- 2016
34. Sensing and Responding to Membrane Tension: The Bacterial MscL Channel as a Model System
- Author
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Irene R Iscla and Paul Blount
- Subjects
Biophysics ,Gating ,Biology ,Mechanotransduction, Cellular ,Models, Biological ,Ion Channels ,Protein Structure, Secondary ,Biophysical Review ,Transmembrane domain ,Protein structure ,Biochemistry ,Bacterial Proteins ,Pressure ,Mechanosensitive channels ,Mechanotransduction ,Lipid bilayer ,Ion channel ,Alpha helix - Abstract
Mechanosensors are important for many life functions, including the senses of touch, balance, and proprioception; cardiovascular regulation; kidney function; and osmoregulation. Many channels from an assortment of families are now candidates for eukaryotic mechanosensors and proprioception, as well as cardiovascular regulation, kidney function, and osmoregulation. Bacteria also possess two families of mechanosensitive channels, termed MscL and MscS, that function as osmotic emergency release valves. Of the two channels, MscL is the most conserved, most streamlined in structure, and largest in conductance at 3.6 nS with a pore diameter in excess of 30 Å; hence, the structural changes required for gating are exaggerated and perhaps more easily defined. Because of these properties, as well as its tractable nature, MscL represents a excellent model for studying how a channel can sense and respond to biophysical changes of a lipid bilayer. Many of the properties of the MscL channel, such as the sensitivity to amphipaths, a helix that runs along the membrane surface and is connected to the pore via a glycine, a twisting and turning of the transmembrane domains upon gating, and the dynamic changes in membrane interactions, may be common to other candidate mechanosensors. Here we review many of these properties and discuss their structural and functional implications.
- Published
- 2012
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35. The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo
- Author
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Paul Blount, Robin Wray, and Irene R Iscla
- Subjects
Pentamer ,fungi ,Conductance ,Biology ,medicine.disease_cause ,Biochemistry ,Membrane ,Tetramer ,Cytoplasm ,Staphylococcus aureus ,Biophysics ,medicine ,Mechanosensitive channels ,Molecular Biology ,Ion channel - Abstract
The mechanosensitive channel of large conductance (MscL) from E. coli serves as an emergency release valve allowing the cell to survive acute osmotic downshock. It is one of the best studied mechanosensitive channels and serves as a paradigm for how a protein can sense and respond to membrane tension. Two MscL crystal structures of the orthologs M. tuberculosis and S. aureus have been solved showing pentameric and tetrameric structures, respectively. Several studies followed to understand whether the discrepancy in their stoichiometry was a species difference or a consequence of the protein manipulation for crystallization. Two independent studies now agree that the full-length S. aureus MscL is actually a pentamer, not tetramer. While detergents appear to play a role in modifying the oligomeric state of the protein, a cytoplasmic helical bundle has also been implicated. Here, we evaluate the role of the C-terminal region of S. aureus MscL in the oligomerization of the channel in native membranes by using an in vivo disulfide-trapping technique. We find that the oligomeric state of S. aureus MscLs with different C-terminal truncations, including the one used to obtain the tetrameric S. aureus MscL crystal structure, are pentamers in vivo. Thus, the C-terminal domain of the S. aureus protein only plays a critical role in the oligomeric state of the SaMscL protein when it is solubilized in detergent.
- Published
- 2011
36. Engineering a pH-Sensitive Liposomal MRI Agent by Modification of a Bacterial Channel
- Author
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S. James Ratnakar, Paul Blount, Quyen N. Do, Bukola Adebesin, Limin Yang, Zoltan Kovacs, and Hui Zheng
- Subjects
0301 basic medicine ,Lysis ,02 engineering and technology ,Article ,Ion Channels ,Biomaterials ,Nanopores ,03 medical and health sciences ,In vivo ,General Materials Science ,Liposome ,Chemistry ,Escherichia coli Proteins ,Vesicle ,General Chemistry ,Hydrogen-Ion Concentration ,021001 nanoscience & nanotechnology ,Magnetic Resonance Imaging ,Kinetics ,Nanopore ,030104 developmental biology ,Membrane ,Targeted drug delivery ,Liposomes ,Biophysics ,Mechanosensitive channels ,0210 nano-technology ,Ion Channel Gating ,Biotechnology - Abstract
MscL is a bacterial mechanosensitive channel that serves as a cellular emergency release valve, protecting the cell from lysis upon a drop in external osmolarity. The channel has an extremely large pore (30 A) and can be purified and reconstituted into artificial membranes. Moreover, MscL is modified to open in response to alternative external stimuli including changes in pH. These properties suggest this channel's potential as a triggered "nanopore" for localized release of vesicular contents such as magnetic resonance imaging (MRI) contrast agents and drugs. Toward this end, several variants of pH-triggered MscL nanovalves are engineered. Stealth vesicles previously been shown to evade normal in vivo clearance and passively accumulate in inflamed and malignant tissues are reconstituted. These vesicles are loaded with 1,4,7,10-tetraazacyclododecane tetraacetic acid gadolinium complex (Gd-DOTA), an MRI contrast reagent, and the resulting nanodevices tested for their ability to release Gd-DOTA as evidenced by enhancement of the longitudinal relaxation rate (R1 ) of the bulk water proton spins. Nanovalves that are responsive to physiological pH changes are identified, but differ in sensitivity and efficacy, thus giving an array of nanovalves that could potentially be useful in different settings. These triggered nanodevices may be useful in delivering both diagnostic and therapeutic agents.
- Published
- 2018
37. Disulfide Trapping the Mechanosensitive Channel MscL into a Gating-Transition State
- Author
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Robin Wray, Gal Levin, Paul Blount, and Irene R Iscla
- Subjects
Models, Molecular ,Patch-Clamp Techniques ,Biophysics ,Gating ,Mechanotransduction, Cellular ,Ion Channels ,Dithiothreitol ,chemistry.chemical_compound ,Osmotic Pressure ,Escherichia coli ,Cysteine ,Disulfides ,Channels, Receptors, and Electrical Signaling ,Ion channel ,Escherichia coli Proteins ,Deletion Mutagenesis ,Transmembrane domain ,Biochemistry ,chemistry ,Mutation ,Mechanosensitive channels ,Dimerization ,Ion Channel Gating ,Oxidation-Reduction ,Linker - Abstract
The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock, and is to date the best characterized mechanosensitive channel. The N-terminal region of the protein has been shown to be critical for function by random, site-directed, and deletion mutagenesis, yet is structurally poorly understood. One model proposes that the extreme N-termini form a cluster of amphipathic helices that serves as a cytoplasmic second gate, separated from the pore-forming transmembrane domain by a “linker”. Here, we have utilized cysteine trapping of single-cysteine mutated channels to determine the proximity, within the homopentameric complex, of residues within and just peripheral to this proposed linker. Our results indicate that all residues in this region can form disulfide bridges, and that the percentage of dimers increases when the channel is gated in vivo. Functional studies suggest that oxidation traps one of these mutated channels, N15C, into a gating-transition state that retains the capacity to obtain both fully open and closed states. The data are not easily explained by current models for the smooth transition from closed-to-open states, but predict that an asymmetric movement of one or more of the subunits commonly occurs upon gating.
- Published
- 2007
38. Mutations in a Conserved Domain of E. coli MscS to the Most Conserved Superfamily Residue Leads to Kinetic Changes
- Author
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Paul Blount and Hannah R. Malcolm
- Subjects
Models, Molecular ,Patch-Clamp Techniques ,Molecular Sequence Data ,Protein domain ,lcsh:Medicine ,Spheroplasts ,Gating ,Biology ,medicine.disease_cause ,Mechanotransduction, Cellular ,Ion Channels ,Protein Structure, Secondary ,Conserved sequence ,Osmotic Pressure ,Escherichia coli ,medicine ,Amino Acid Sequence ,lcsh:Science ,Conserved Sequence ,Ion channel ,Mutation ,Ion Transport ,Multidisciplinary ,Sequence Homology, Amino Acid ,Mechanosensation ,Escherichia coli Proteins ,lcsh:R ,Gene Expression Regulation, Bacterial ,Protein Structure, Tertiary ,Cell biology ,Kinetics ,Biochemistry ,Mechanosensitive channels ,lcsh:Q ,Ion Channel Gating ,Research Article - Abstract
In Escherichia coli (E. coli) the mechanosensitive channel of small conductance, MscS, gates in response to membrane tension created from acute external hypoosmotic shock, thus rescuing the bacterium from cell lysis. E. coli MscS is the most well studied member of the MscS superfamily of channels, whose members are found throughout the bacterial and plant kingdoms. Homology to the pore lining helix and upper vestibule domain of E. coli MscS is required for inclusion into the superfamily. Although highly conserved, in the second half of the pore lining helix (TM3B), E. coli MscS has five residues significantly different from other members of the superfamily. In superfamilies such as this, it remains unclear why variations within such a homologous region occur: is it tolerance of alternate residues, or does it define functional variance within the superfamily? Point mutations (S114I/T, L118F, A120S, L123F, F127E/K/T) and patch clamp electrophysiology were used to study the effect of changing these residues in E. coli MscS on sensitivity and gating. The data indicate that variation at these locations do not consistently lead to wildtype channel phenotypes, nor do they define large changes in mechanosensation, but often appear to effect changes in the E. coli MscS channel gating kinetics.
- Published
- 2015
39. Mechanosensitive Channel Gating Transitions Resolved by Functional Changes upon Pore Modification
- Author
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Paul Blount, Yuezhou Li, and Jessica L. Bartlett
- Subjects
Models, Molecular ,Chemistry ,Protein Conformation ,Escherichia coli Proteins ,Analytical chemistry ,Biophysics ,Conductance ,Gating ,Mechanotransduction, Cellular ,Models, Biological ,Ion Channels ,Molecular dynamics ,Structure-Activity Relationship ,Protein structure ,Models, Chemical ,Sulfhydryl reagent ,Mechanosensitive channels ,Computer Simulation ,Channels, Receptors, and Electrical Signaling ,Ion Channel Gating ,Porosity ,Ion channel ,Communication channel - Abstract
The mechanosensitive channel of large conductance acts as a biological “emergency release valve” that protects bacterial cells from hypoosmotic stress. Although structural and functional studies and molecular dynamic simulations of this channel have led to several models for the structural transitions that occur in the gating process, inconsistencies linger and details are lacking. A previous study, using a method coined as the “in vivo SCAM”, identified several residues in the channel pore that were exposed to the aqueous environment in the closed and opening conformations. Briefly, the sulfhydryl reagent MTSET was allowed to react, in the presence or absence of hypoosmotic shock, with cells expressing mechanosensitive channel of large conductance channels that contained cysteine substitutions; channel dysfunction was assessed solely by cell viability. Here we evaluate the MTSET-induced functional modifications to these mechanosensitive channel activities by measuring single channel recordings. The observed changes in residue availability in different states, as well as channel kinetics and sensitivity, have allowed us to elucidate the microenvironment encountered for a number of pore residues, thus testing many aspects of previous models and giving a higher resolution of the pore domain and the structural transitions it undergoes from the closed to open state.
- Published
- 2006
- Full Text
- View/download PDF
40. Assessment of Potential Stimuli for Mechano-Dependent Gating of MscL: Effects of Pressure, Tension, and Lipid Headgroups
- Author
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Paul Blount and Paul C. Moe
- Subjects
Patch-Clamp Techniques ,Chemistry ,Escherichia coli Proteins ,Cell Membrane ,fungi ,Gating ,Lipids ,Biochemistry ,Ion Channels ,Electrophysiology ,Cell membrane ,medicine.anatomical_structure ,Membrane ,Membrane curvature ,Escherichia coli ,Pressure ,medicine ,Biophysics ,Mechanosensitive channels ,Lipid bilayer ,Ion Channel Gating ,Ion channel ,Elasticity of cell membranes - Abstract
MscL is a mechanosensitive channel of large conductance that serves as an "emergency relief valve", protecting bacteria from acute hypoosmotic stress. Although it is well-accepted that the MscL protein and an adequate membrane matrix are necessary and sufficient for the function of this channel, the exact role of the membrane has yet to be elucidated. Here, we address the role of the membrane matrix through in vitro reconstitution of the MscL protein in defined lipid bilayers. We have applied Laplace's law to visualized membrane patches where we can measure patch curvature as described in previous studies. Here, by comparing patches with different curvatures, we demonstrate that the MscL channel senses tension within the membrane and that the pressure across it plays no detectable role as a stimulus. In addition, gating only occurs when the smallest radius of curvature is nearly achieved, suggesting that the lateral tension rather than membrane curvature is the important biophysical parameter. Finally, we have examined the contribution of specific headgroups by measuring their effect on the membrane tension required to gate the channel. We have found that the addition of neither anionic nor endogenous lipids to a non-native membrane effected a leftward shift in the activation curve. In fact, the major endogenous lipid of the Escherichia coli membrane, phosphatidylethanolamine, led to a channel activity at a higher tension threshold, suggesting that this lipid effects altered activity through changes in the biophysical properties of the membrane, rather than through an MscL-specific interaction.
- Published
- 2005
41. Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel
- Author
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Irene R Iscla, Sally Dennison, Yuezhou Li, Susan Shirley Black, Wendy Bartlett, Sanguk Kim, Paul Blount, Ian R. Booth, Michelle D. Edwards, Samantha Miller, and James U. Bowie
- Subjects
Models, Molecular ,Alanine ,Escherichia coli Proteins ,Glycine ,DNA replication ,Gating ,Protein degradation ,Biology ,Ion Channels ,Protein Structure, Tertiary ,Cell biology ,Electrophysiology ,Transmembrane domain ,Phenotype ,Structural Biology ,Mutation ,Helix ,Escherichia coli ,Protein folding ,Mechanosensitive channels ,Molecular Biology - Abstract
The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was recently solved. However, the conformation of the closed state and the gating transition remain uncharacterized. The pore-lining transmembrane helix contains a conserved glycine- and alanine-rich motif that forms a helix-helix interface. We show that introducing 'knobs' on the smooth glycine face by replacing glycine with alanine, and substituting conserved alanines with larger residues, increases the pressure required for gating. Creation of a glycine-glycine interface lowers activation pressure. The importance of residues Gly104, Ala106 and Gly108, which flank the hydrophobic seal, is demonstrated. A new structural model is proposed for the closed-to-open transition that involves rotation and tilt of the pore-lining helices. Introduction of glycine at Ala106 validated this model by acting as a powerful suppressor of defects seen with mutations at Gly104 and Gly108.
- Published
- 2005
42. Lactococcus lactis Uses MscL as Its Principal Mechanosensitive Channel
- Author
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Paul Blount, Joost H.A. Folgering, Berend Poolman, Gea K. Schuurman-Wolters, Paul C. Moe, Groningen Biomolecular Sciences and Biotechnology, Biomonitoring and Sensoring, Enzymology, Faculty of Science and Engineering, and Zernike Institute for Advanced Materials
- Subjects
ESCHERICHIA-COLI-CELLS ,Patch-Clamp Techniques ,Mutant ,Molecular Sequence Data ,STREPTOCOCCUS-LACTIS ,medicine.disease_cause ,Aquaporins ,Biochemistry ,GLYCINE BETAINE ,03 medical and health sciences ,chemistry.chemical_compound ,Betaine ,medicine ,Amino Acid Sequence ,Molecular Biology ,Escherichia coli ,IN-VIVO ,030304 developmental biology ,DNA Primers ,0303 health sciences ,Osmotic concentration ,biology ,Base Sequence ,Sequence Homology, Amino Acid ,030306 microbiology ,MEMBRANE-PROTEINS ,Lactococcus lactis ,Cell Membrane ,fungi ,Wild type ,SINGLE RESIDUE ,Cell Biology ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,CONTROLLED GENE-EXPRESSION ,TRANSPORT ,Cell biology ,chemistry ,Amino Acid Substitution ,Glycine ,Mutagenesis, Site-Directed ,LARGE-CONDUCTANCE ,Cystine ,Mechanosensitive channels ,Calcium Channels ,MYCOBACTERIUM-TUBERCULOSIS ,Sequence Alignment - Abstract
The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.
- Published
- 2005
43. Intragenic suppression of gain-of-function mutations in the Escherichia coli mechanosensitive channel, MscL
- Author
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Yuezhou Li, Robin Wray, and Paul Blount
- Subjects
Genetics ,Mutation ,Mutant ,Mutagenesis (molecular biology technique) ,Periplasmic space ,Biology ,medicine.disease_cause ,Microbiology ,Transmembrane protein ,Cell biology ,S1 domain ,Transmembrane domain ,medicine ,Mechanosensitive channels ,Molecular Biology - Abstract
Mechanosensitive channels play an important role in protecting bacterial cells from osmotic downshock by serving as biological 'pressure release valves'. One of these channels, MscL, is found throughout the bacterial kingdom, but has been most studied in Escherichia coli. The E. coli MscL is a 136-amino-acid protein organized as a homopentamer with each subunit containing two transmembrane segments. Previous studies have shown that several residues, including V23 and G26, are essential for normal function of MscL; very severe gain-of-function phenotypes in which cell growth slows or is arrested can result from residue substitutions at these positions. Through random mutagenesis and growth selection, we have generated intragenic suppressors of the V23A and G26S mutations. The suppressor mutants have been characterized by growth phenotype, Western blot and patch clamp. Most of the mutations that render phenotypic suppression are located in the transmembrane domains with additional sites lying in the periplasmic loop. In contrast, only one mutation is found in the amino-terminal S1 domain, and none is found within the carboxyl-terminal domain. Not only have these findings revealed functional domains and subdomains critical for MscL function, but they also predict a pair of residues that interact directly during channel opening.
- Published
- 2004
44. Channels in microbes: so many holes to fill
- Author
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Paul Blount and Ching Kung
- Subjects
Communication ,business.industry ,technology, industry, and agriculture ,Biology ,business ,Molecular Biology ,Microbiology ,humanities ,Ion channel ,Microbial Physiology ,Communication channel - Abstract
Among players in neurobiology, ion channels are the demigods that underlie all our senses, behaviour and intelligence. In animals, these 'gated pores' detect ligands, voltage, heat or stretch forces and emit electric or ionic signals. Patch clamp and genome sequencing now show that nearly all microbes also have these 'smart' molecules. Microbial channel proteins have yielded crystal structures so dear to neuroscientists. However, their natural roles in microbial physiology remain largely unknown. The intellectual and technical schisms between 'neuro' and 'micro' biology must be bridged before we know how we became so smart, and whether microbes are just as smart.
- Published
- 2004
45. Cysteine Scanning of MscL Transmembrane Domains Reveals Residues Critical for Mechanosensitive Channel Gating
- Author
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Gal Levin and Paul Blount
- Subjects
Models, Molecular ,Osmosis ,Protein Conformation ,Blotting, Western ,Mutant ,Biophysics ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Ion Channels ,Protein structure ,Channels, Receptors, and Transporters ,Escherichia coli ,medicine ,Cysteine ,Disulfides ,Ion channel ,Mutation ,Escherichia coli Proteins ,Mutagenesis ,Mycobacterium tuberculosis ,Protein Structure, Tertiary ,Electrophysiology ,Kinetics ,Transmembrane domain ,Phenotype ,Biochemistry ,Mutagenesis, Site-Directed ,Mechanosensitive channels - Abstract
The mechanosensitive channel of large conductance (MscL), a bacterial channel, is perhaps the best characterized mechanosensitive protein. A structure of the Mycobacterium tuberculosis ortholog has been solved by x-ray crystallography, but details of how the channel gates remain obscure. Here, cysteine scanning was used to identify residues within the transmembrane domains of Escherichia coli MscL that are crucial for normal function. Utilizing genetic screens, we identified several mutations that induced gain-of-function or loss-of-function phenotypes in vivo. Mutants that exhibited the most severe phenotypes were further characterized using electrophysiological techniques and chemical modifications of the substituted cysteines. Our results verify the importance of residues in the putative primary gate in the first transmembrane domain, corroborate other residues previously noted as critical for normal function, and identify new ones. In addition, evaluation of disulfide bridging in native membranes suggests alterations of existing structural models for the “fully closed” state of the channel.
- Published
- 2004
46. A Lipid-Exposed Residue at the Start of S4-S5 Linker Controls TRPV4 Gating
- Author
-
Andriy Anishkin, Ching Kung, Paul Blount, Jinfeng Teng, and Stephen H. Loukin
- Subjects
chemistry.chemical_classification ,Residue (chemistry) ,Transient receptor potential channel ,Hydrogen bond ,Chemistry ,Stereochemistry ,Biophysics ,Side chain ,Gating ,Linker ,Ion channel ,Amino acid - Abstract
We have some generalized physical understanding of how ion channels interact with surrounding lipids but lack detailed descriptions on how interactions of particular amino acid with contacting lipids may regulate gating. Here we show a structure-specific interaction between an amino acid and inner-leaflet lipid that governs the gating transformations of TRPV4 (Transient Receptor Potential Vanilloid type 4). Many cation channels use a S4-S5 linker to transmit stimuli to the gate. At the start of TRPV4's linker helix is leucine 596. A hydrogen bond between L596's backbone oxygen and the indole of W733 of the TRP helix acts as a latch to maintain channel closure. The side chain of L596 interacts with the inner lipid leaflet near the polar-nonpolar interface in our model - an interaction that we explored by mutagenesis. We examined the outward currents of TRPV4-expressing Xenopus oocyte upon depolarizations as well as phenotypes of expressing yeast cells. Maintaining its hydrophobicity, L596F and L596V channels appear normal in gating. However, making this residue less hydrophobic (L596A, G, W, Q, K) reduces open probability (Po) (loss-of-function, LOF), likely due to altered interactions at the polar-nonpolar interface. L596I raises Po (gain-of-function, GOF), apparently by placing its methyl group closer to the polar region and receiving stronger water repulsion. Molecular dynamics simulations showed that the distance between the β carbons of H-bonded residues 596 and 733 is shortened in the LOFs and lengthened in the GOFs, strengthening or weakening the linker/TRP-helix latch respectively. These results highlight that, in each lipid-exposed focus, the L596-lipid attraction counteracts the latch bond in a tug-of-war to tune the Po of TRPV4.
- Published
- 2016
47. Molecular Mechanisms of Mechanosensation
- Author
-
Paul Blount
- Subjects
Mechanosensation ,Membrane protein ,General Neuroscience ,Neuroscience(all) ,Mutant ,Mechanosensitive channels ,Gating ,Biology ,Cell biology - Abstract
Little is known of molecular mechanisms of human mechanosensation. Only now are candidate eukaryotic sensors being identified. In contrast, bacterial sensors, including mechanosensitive channels, have been cloned, sequenced, reconstituted, and functional mutants characterized. Moreover, crystal structures for bacterial mechanosensitive channels have been resolved and structural gating transitions predicted. These studies give clues to general principles underlying the ability of a membrane protein to sense and respond to perturbations of its lipid environment that may be conserved between bacteria and humans.
- Published
- 2003
- Full Text
- View/download PDF
48. Family ties of gated pores: evolution of the sensor module
- Author
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Gal Levin, Paul Blount, and Attila Kumánovics
- Subjects
Models, Molecular ,Family ties ,Molecular Sequence Data ,Computational biology ,Biology ,Mechanotransduction, Cellular ,Biochemistry ,Ion Channels ,Evolution, Molecular ,Transient receptor potential channel ,Bacterial Proteins ,Genetics ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Sequence Homology, Amino Acid ,Voltage-gated ion channel ,business.industry ,Modular design ,Phenotype ,Transmembrane domain ,Mechanosensitive channels ,business ,Ion Channel Gating ,Biotechnology ,Communication channel - Abstract
The six-transmembrane channels are thought to be composed of two modules: pore and sensor. Whereas the modular design of the pore has been established, the modularity of the sensor remains hypothetical. As a first step toward establishing the modularity of this region, we searched for genes where the sensor is found independent of the pore and have identified new members of the sensor superfamily. Analysis of these sensors reveals a motif shared among not only these newly discovered members and voltage-gated, transient receptor potential, and polycystin channel sensors, but also MscL, a bacterial mechanosensitive channel. Mutational analyses presented here and in previous studies demonstrate that highly conserved residues within this motif are required for normal channel activity; mutations of residues within this motif in different subfamilies lead to consistent channel phenotypes. Previous studies have demonstrated that peptides containing this motif and the adjacent conserved transmembrane domain elicit channel activities when reconstituted into lipid membranes. These data provide evidence for the modularity of the sensor, imply a model for its evolution, suggest a common origin for mechano- and voltage-sensing, and may offer a glimpse of the properties of the first sensor/channel.
- Published
- 2002
49. How do membrane proteins sense water stress?
- Author
-
Paul Blount, R.H.E. Friesen, Berend Poolman, Paul C. Moe, Tiemen van der Heide, and Joost H.A. Folgering
- Subjects
Membrane protein ,Biochemistry ,Osmotic shock ,Turgor pressure ,Biophysics ,Osmotic pressure ,Mechanosensitive channels ,Osmoprotectant ,Biology ,Cell envelope ,Molecular Biology ,Microbiology ,Intracellular - Abstract
Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmoprotectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein-lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.
- Published
- 2002
50. The mechanosensitive channel of small conductance (MscS) functions as a Jack-in-the box
- Author
-
Paul Blount, Joshua A. Maurer, and Hannah R. Malcolm
- Subjects
Models, Molecular ,Patch-Clamp Techniques ,Lipid Bilayers ,Biophysics ,Gating ,Spheroplasts ,Biochemistry ,Mechanotransduction, Cellular ,Ion Channels ,Article ,Membrane Potentials ,Jack-In-The-Box ,Escherichia coli ,Pressure ,Lipid bilayer ,Gating mechanism ,Ion channel ,Alanine ,Membrane potential ,Mechanosensitive channel of small conductance (MscS) ,Lipid interaction ,Binding Sites ,Chemistry ,Bilayer ,Escherichia coli Proteins ,Cell Biology ,Protein Structure, Tertiary ,Transmembrane domain ,Mutation ,Mechanosensitive channels ,Bacterial ion channel ,Ion Channel Gating ,Protein Binding - Abstract
Phenotypical analysis of the lipid interacting residues in the closed state of the mechanosensitive channel of small conductance (MscS) from Escherichia coli (E. coli) has previously shown that these residues are critical for channel function. In the closed state, mutation of individual hydrophobic lipid lining residues to alanine, thus reducing the hydrophobicity, resulted in phenotypic changes that were observable using in vivo assays. Here, in an analogous set of experiments, we identify eleven residues in the first transmembrane domain of the open state of MscS that interact with the lipid bilayer. Each of these residues was mutated to alanine and leucine to modulate their hydrophobic interaction with the lipid tail-groups in the open state. The effects of these changes on channel function were analyzed using in vivo bacterial assays and patch clamp electrophysiology. Mutant channels were found to be functionally indistinguishable from wildtype MscS. Thus, mutation of open-state lipid interacting residues does not differentially stabilize or destabilize the open, closed, intermediate, or transition states of MscS. Based on these results and other data from the literature, we propose a new gating paradigm for MscS where MscS acts as a “Jack-In-The-Box” with the intrinsic bilayer lateral pressure holding the channel in the closed state. In this model, upon application of extrinsic tension the channel springs into the open state due to relief of the intrinsic lipid bilayer pressure.
- Published
- 2014
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