faye blue | Ashford University (original) (raw)
Papers by faye blue
Current Opinion in Microbiology, 2004
Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the... more Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the millisecond timescale they must achieve the transition from tightly closed oligomers to large, relatively non-discriminating pores. The crystal structure for MscL, combined with genetic and biochemical analysis, provided the initial insights for the mechanism by which this structural transition might be made. Discovery of the gene for a second class of mechanosensitive channel, MscS, and its subsequent crystallisation, has provided a new paradigm for mechanosensation, enabling a deeper understanding of the mechanisms of sensing membrane tension.
Nature Reviews Microbiology, 2007
| Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer ... more | Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.
Structure, 2009
KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of ... more KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of various K + channels and transporters. The mechanisms these proteins use to control their transmembrane pore-forming counterparts remains unclear despite numerous electrophysiological and structural studies. KTN (RCK) domains consistently crystallize as dimers within the asymmetric unit, forming a pronounced hinge between two Rossmann folds. We have previously proposed that modification of the hinge angle plays an important role in activating the associated membraneintegrated components of the channel or transporter. Here we report the structure of the C-terminal, KTN-bearing domain of the E. coli KefC K + efflux system in association with the ancillary subunit, KefF, which is known to stabilize the conductive state. The structure of the complex and functional analysis of KefC variants reveal that control of the conformational flexibility inherent in the KTN dimer hinge is modulated by KefF and essential for regulation of KefC ion flux.
Current Topics in Membranes, 2007
The mechanosensitive (MS) channel MscS is the more widespread of two major MS channels that have ... more The mechanosensitive (MS) channel MscS is the more widespread of two major MS channels that have been characterized. MscS‐like proteins have been discovered in bacteria, archaea, yeasts and fungi, and in plants. In most organisms, multiple ...
Biochemistry, 2003
This is one of the most exciting periods in membrane biology. The last five years has seen the el... more This is one of the most exciting periods in membrane biology. The last five years has seen the elucidation of the structures of several bacterial ion channels (1− 6). These structures have allowed central questions about the mechanism (s) of selectivity and gating of ion ...
Methods in Enzymology, 2007
Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypo... more Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypoosmotic shock. Bacteria that have been diluted from high osmolarity medium into dilute solution are required to cope with sudden water influx associated with an osmotic imbalance equivalent to 10 to 14 atm. The cell wall is only poorly expansive and the cytoplasmic membrane even less so. Thus, swelling is not an option and the cell must rapidly eject solutes to diminish the osmotic gradient and thereby preserve structural integrity. This chapter describes cellular assays of MS channel function and their interpretation.
Nature Structural & Molecular Biology, 2005
The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was r... more 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.
Microbiology and Molecular Biology Reviews, 2003
Science, 2008
How ion channels are gated to regulate ion flux in and out of cells is the subject of intense int... more How ion channels are gated to regulate ion flux in and out of cells is the subject of intense interest. The Escherichia coli mechanosensitive channel, MscS, opens to allow rapid ion efflux, relieving the turgor pressure that would otherwise destroy the cell. We present a 3.45 angstrom-resolution structure for the MscS channel in an open conformation. This structure has a pore diameter of 13 angstroms created by substantial rotational rearrangement of the three transmembrane helices. The structure suggests a molecular mechanism that underlies MscS gating and its decay of conductivity during prolonged activation. Support for this mechanism is provided by single-channel analysis of mutants with altered gating characteristics.
Biochemistry, 2007
Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched ... more Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with phenylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability.
Current Opinion in Microbiology, 2004
Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the... more Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the millisecond timescale they must achieve the transition from tightly closed oligomers to large, relatively non-discriminating pores. The crystal structure for MscL, combined with genetic and biochemical analysis, provided the initial insights for the mechanism by which this structural transition might be made. Discovery of the gene for a second class of mechanosensitive channel, MscS, and its subsequent crystallisation, has provided a new paradigm for mechanosensation, enabling a deeper understanding of the mechanisms of sensing membrane tension.
Nature Reviews Microbiology, 2007
| Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer ... more | Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.
Structure, 2009
KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of ... more KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of various K+ channels and transporters. The mechanisms these proteins use to control their transmembrane pore-forming counterparts remains unclear despite numerous electrophysiological and structural studies. KTN (RCK) domains consistently crystallize as dimers within the asymmetric unit, forming a pronounced hinge between two Rossmann folds. We have previously proposed that modification of the hinge angle plays an important role in activating the associated membrane-integrated components of the channel or transporter. Here we report the structure of the C-terminal, KTN-bearing domain of the E. coli KefC K+ efflux system in association with the ancillary subunit, KefF, which is known to stabilize the conductive state. The structure of the complex and functional analysis of KefC variants reveal that control of the conformational flexibility inherent in the KTN dimer hinge is modulated by KefF and essential for regulation of KefC ion flux.
Current Topics in Membranes, 2007
The mechanosensitive (MS) channel MscS is the more widespread of two major MS channels that have ... more The mechanosensitive (MS) channel MscS is the more widespread of two major MS channels that have been characterized. MscS‐like proteins have been discovered in bacteria, archaea, yeasts and fungi, and in plants. In most organisms, multiple ...
Biochemistry, 2003
This is one of the most exciting periods in membrane biology. The last five years has seen the el... more This is one of the most exciting periods in membrane biology. The last five years has seen the elucidation of the structures of several bacterial ion channels (1− 6). These structures have allowed central questions about the mechanism (s) of selectivity and gating of ion ...
Methods in Enzymology, 2007
Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypo... more Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypoosmotic shock. Bacteria that have been diluted from high osmolarity medium into dilute solution are required to cope with sudden water influx associated with an osmotic imbalance equivalent to 10 to 14 atm. The cell wall is only poorly expansive and the cytoplasmic membrane even less so. Thus, swelling is not an option and the cell must rapidly eject solutes to diminish the osmotic gradient and thereby preserve structural integrity. This chapter describes cellular assays of MS channel function and their interpretation.
Nature Structural & Molecular Biology, 2005
The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was r... more 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.
Microbiology and Molecular Biology Reviews, 2003
Current Opinion in Microbiology, 2004
Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the... more Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the millisecond timescale they must achieve the transition from tightly closed oligomers to large, relatively non-discriminating pores. The crystal structure for MscL, combined with genetic and biochemical analysis, provided the initial insights for the mechanism by which this structural transition might be made. Discovery of the gene for a second class of mechanosensitive channel, MscS, and its subsequent crystallisation, has provided a new paradigm for mechanosensation, enabling a deeper understanding of the mechanisms of sensing membrane tension.
Nature Reviews Microbiology, 2007
| Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer ... more | Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.
Structure, 2009
KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of ... more KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of various K + channels and transporters. The mechanisms these proteins use to control their transmembrane pore-forming counterparts remains unclear despite numerous electrophysiological and structural studies. KTN (RCK) domains consistently crystallize as dimers within the asymmetric unit, forming a pronounced hinge between two Rossmann folds. We have previously proposed that modification of the hinge angle plays an important role in activating the associated membraneintegrated components of the channel or transporter. Here we report the structure of the C-terminal, KTN-bearing domain of the E. coli KefC K + efflux system in association with the ancillary subunit, KefF, which is known to stabilize the conductive state. The structure of the complex and functional analysis of KefC variants reveal that control of the conformational flexibility inherent in the KTN dimer hinge is modulated by KefF and essential for regulation of KefC ion flux.
Current Topics in Membranes, 2007
The mechanosensitive (MS) channel MscS is the more widespread of two major MS channels that have ... more The mechanosensitive (MS) channel MscS is the more widespread of two major MS channels that have been characterized. MscS‐like proteins have been discovered in bacteria, archaea, yeasts and fungi, and in plants. In most organisms, multiple ...
Biochemistry, 2003
This is one of the most exciting periods in membrane biology. The last five years has seen the el... more This is one of the most exciting periods in membrane biology. The last five years has seen the elucidation of the structures of several bacterial ion channels (1− 6). These structures have allowed central questions about the mechanism (s) of selectivity and gating of ion ...
Methods in Enzymology, 2007
Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypo... more Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypoosmotic shock. Bacteria that have been diluted from high osmolarity medium into dilute solution are required to cope with sudden water influx associated with an osmotic imbalance equivalent to 10 to 14 atm. The cell wall is only poorly expansive and the cytoplasmic membrane even less so. Thus, swelling is not an option and the cell must rapidly eject solutes to diminish the osmotic gradient and thereby preserve structural integrity. This chapter describes cellular assays of MS channel function and their interpretation.
Nature Structural & Molecular Biology, 2005
The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was r... more 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.
Microbiology and Molecular Biology Reviews, 2003
Science, 2008
How ion channels are gated to regulate ion flux in and out of cells is the subject of intense int... more How ion channels are gated to regulate ion flux in and out of cells is the subject of intense interest. The Escherichia coli mechanosensitive channel, MscS, opens to allow rapid ion efflux, relieving the turgor pressure that would otherwise destroy the cell. We present a 3.45 angstrom-resolution structure for the MscS channel in an open conformation. This structure has a pore diameter of 13 angstroms created by substantial rotational rearrangement of the three transmembrane helices. The structure suggests a molecular mechanism that underlies MscS gating and its decay of conductivity during prolonged activation. Support for this mechanism is provided by single-channel analysis of mutants with altered gating characteristics.
Biochemistry, 2007
Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched ... more Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with phenylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability.
Current Opinion in Microbiology, 2004
Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the... more Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the millisecond timescale they must achieve the transition from tightly closed oligomers to large, relatively non-discriminating pores. The crystal structure for MscL, combined with genetic and biochemical analysis, provided the initial insights for the mechanism by which this structural transition might be made. Discovery of the gene for a second class of mechanosensitive channel, MscS, and its subsequent crystallisation, has provided a new paradigm for mechanosensation, enabling a deeper understanding of the mechanisms of sensing membrane tension.
Nature Reviews Microbiology, 2007
| Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer ... more | Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.
Structure, 2009
KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of ... more KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of various K+ channels and transporters. The mechanisms these proteins use to control their transmembrane pore-forming counterparts remains unclear despite numerous electrophysiological and structural studies. KTN (RCK) domains consistently crystallize as dimers within the asymmetric unit, forming a pronounced hinge between two Rossmann folds. We have previously proposed that modification of the hinge angle plays an important role in activating the associated membrane-integrated components of the channel or transporter. Here we report the structure of the C-terminal, KTN-bearing domain of the E. coli KefC K+ efflux system in association with the ancillary subunit, KefF, which is known to stabilize the conductive state. The structure of the complex and functional analysis of KefC variants reveal that control of the conformational flexibility inherent in the KTN dimer hinge is modulated by KefF and essential for regulation of KefC ion flux.
Current Topics in Membranes, 2007
The mechanosensitive (MS) channel MscS is the more widespread of two major MS channels that have ... more The mechanosensitive (MS) channel MscS is the more widespread of two major MS channels that have been characterized. MscS‐like proteins have been discovered in bacteria, archaea, yeasts and fungi, and in plants. In most organisms, multiple ...
Biochemistry, 2003
This is one of the most exciting periods in membrane biology. The last five years has seen the el... more This is one of the most exciting periods in membrane biology. The last five years has seen the elucidation of the structures of several bacterial ion channels (1− 6). These structures have allowed central questions about the mechanism (s) of selectivity and gating of ion ...
Methods in Enzymology, 2007
Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypo... more Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypoosmotic shock. Bacteria that have been diluted from high osmolarity medium into dilute solution are required to cope with sudden water influx associated with an osmotic imbalance equivalent to 10 to 14 atm. The cell wall is only poorly expansive and the cytoplasmic membrane even less so. Thus, swelling is not an option and the cell must rapidly eject solutes to diminish the osmotic gradient and thereby preserve structural integrity. This chapter describes cellular assays of MS channel function and their interpretation.
Nature Structural & Molecular Biology, 2005
The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was r... more 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.
Microbiology and Molecular Biology Reviews, 2003