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H Bayley

Publications and source records attributed to H Bayley.

At least 55 records · Page 3Linked to original sources
PubMed1997

Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel.

Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacterial exotoxin, which forms heptameric pores in membranes. The rate determining step in assembly is the conversion of a heptameric prepore to the fully assembled pore in which the central glycine-rich domain of each subunit inserts into the membrane to form a 14 strand beta barrel. Barrel formation is accompanied by a conformational change in which each N terminus latches onto an adjacent subunit. In the monomer in solution, the central domain is loosely organized and exposed to solvent. In this study, 25 amino acids of the central domain were removed and replaced with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity. Within minutes after synthesis in the absence of membranes, the mutant polypeptide spontaneously assembled into heptamers, as demonstrated by atomic force microscopy. Limited proteolysis suggested that the N termini of the subunits in the heptamers were in the fully assembled pore conformation rather than the prepore conformation. Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces the additional structural changes that normally accompany pore formation. The truncated pore might make a useful framework for the construction of designed membrane active macromolecules.

Amino Acid Sequence↗
PubMed1997

Applications of S-layers.

The wealth of information existing on the general principle of S-layers has revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from many organisms are capable of recrystallizing as closed monolayers onto solid supports at the air-water interface, on lipid monolayers or onto the surface of liposomes. Particularly their repetitive physicochemical properties down to the subnanometer scale make S-layers unique structures for functionalization of surfaces and interfaces down to the ultimate resolution limit. The following review focuses on selected applications in biotechnology, diagnostics, vaccine development, biomimetic membranes, supramolecular engineering and nanotechnology. Despite progress in the characterization of S-layers and the exploitation of S-layers for the applications described in this chapter, it is clear that the field lags behind others (e.g. enzyme engineering) in applying recent advances in protein engineering. Genetic modification and targeted chemical modification would allow several possibilities including the manipulation of pore permeation properties, the introduction of switches to open and close the pores, and the covalent attachment to surfaces or other macromolecules through defined sites on the S-layer protein. The application of protein engineering to S-layers will require the development of straightforward expression systems, the development of simple assays for assembly and function that are suitable for the rapid screening of numerous mutants and the acquisition of structural information at atomic resolution. Attention should be given to these areas in the coming years.

Bacteria↗
PubMed1996

Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore.

The structure of the Staphylococcus aureus alpha-hemolysin pore has been determined to 1.9 A resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 A in length, that runs along the sevenfold axis and ranges from 14 A to 46 A in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel beta barrel, to which each protomer contributes two beta strands, each 65 A long. The interior of the beta barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 A wide. The structure proves the heptameric subunit stoichiometry of the alpha-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of beta barrel pore-forming toxins.

Amino Acid Sequence↗
PubMed1996

Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin.

Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre-pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically labelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally distinct parts could be identified. For mutants at positions 126-140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the membrane lipids. Membrane insertion of this sequence occurred together with the final pre-pore to pore transition of the heptamer. Thus residues 126-140 constitute a transmembrane sequence in the pore. With labelled residues 118-124, pre-pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer-protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore.

2-Naphthylamine↗
PubMed1996

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins.

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin are well-studied prototypes of pore-forming bacterial cytotoxins. Each is produced as a water-soluble single-chain polypeptide that inserts into target membranes to form aqueous transmembrane pores. This review will compare properties of the three toxin prototypes, highlighting the similarities and also the differences in their structure, mode of binding, mechanism of pore formation, and the responses they elicit in target cells. Pore-forming toxins represent the most potent and versatile weapons with which invading microbes damage the host macroorganism.

Amino Acid Sequence↗
PubMed1996

Alternative splicing of the NMDAR1 subunit affects modulation by calcium.

Four splice variants of the NR1 receptor subunit, characterized by the presence or absence of cassettes encoding inserts of 21 (Insert 1) and 37 (Insert 2) amino acids were expressed in Xenopus oocytes and studied using voltage-clamp techniques. In 1.8 mM Ca2+, a slow inward current (Islow), which peaked 20 s after exposure to NMDA was evident when Insert I was present, but not when absent. However, in elevated external Ca2+ medium a similar Islow was observed in variants missing Insert I. The Ca2+ dependency of Islow reflected a requirement for intracellular accumulation of Ca2+. The divalent ion permeability of Insert I containing and Insert 1 lacking receptor channels expressed alone, as well as in heteromeric assemblies with NR2A and NR2B, was similar for all combinations tested. Thus, the lower Ca2+ dependency for Islow in oocytes expressing Insert I was not due to higher calcium entry. Islow was less sensitive to blockers of ICl(Ca) than were endogenous calcium-activated chloride currents (ICl(Ca)). Also, Islow was not abolished in Cl(-)-free external medium, when voltage was manipulated such that Islow was outward-going. Thus, Islow, while containing a component due to activation of endogenous ICl(Ca), is primarily due to current flowing through the receptor ion channel. Development of Islow was unaffected by PKC or PKA inhibitors. The modulation of the Ca2+ dependency of Islow by Insert I occurs in a range of Ca2+ concentrations which are physiologically relevant, and may provide an important means of modulation of glutamate transmission under normal and pathological conditions.

Alternative Splicing↗
PubMed1996

Tumor protease-activated, pore-forming toxins from a combinatorial library.

We describe a library of two-chain molecular complementation mutants of staphylococcal alpha-hemolysin that features a combinatorial cassette encoding thousands of protease recognition sites in the central pore-forming domain. The cassette is flanked by a peptide extension that inactivates the protein. We screened the library to identify alpha-hemolysins that are highly susceptible to activation by cathepsin B, a protease that is secreted by certain metastatic tumor cells. Toxins obtained by this procedure should be useful for the permeabilization of malignant cells thereby leading directly to cell death or permitting destruction of the cells with drugs that are normally membrane impermeant.

Amino Acid Sequence↗
PubMed1996

Inhibitory effects of ketamine and halothane on recombinant potassium channels from mammalian brain.

BACKGROUND: There is increasing evidence that direct interactions between volatile anesthetics and channel proteins may result in general anesthesia. Using voltage-clamp techniques, the authors examined the effect of two general anesthetics (ketamine and halothane) on a rat brain potassium channel of known amino acid sequence, and further assessed whether the inhibition of the channel is altered by a partial deletion of the C-terminal sequence of this channel. METHODS: Xenopus laevis oocytes were microinjected with either Kv2.1 or delta C318 (a mutated channel in which the last 318 amino acids of the C-terminus have been deleted) cRNA, and channel function in translated channels was observed before, during, and after exposure to graded concentrations of ketamine (25, 50, and 75 micrometers) and halothane (1%, 2%, and 4%). RESULTS: Ketamine and halothane reduced Kv2.1 and delta C318 peak current amplitude in a dose-dependent and reversible fashion. The inhibition of current was voltage dependent for halothane but not for ketamine. Halothane accelerated the time constant of current inactivation, whereas ketamine affected this parameter minimally in both channel types. Use dependence of ketamine and halothane action was observed in both Kv2.1 and the mutant channel, attributable to augmentation of C-type inactivation. CONCLUSIONS: Although both ketamine and halothane inhibit potassium currents through the Kv2.1 channel, their mechanisms of action at this potential target may be different. Deletion of the C-terminal sequence resulted in decreased sensitivity to both anesthetics. Although it is not clear whether anesthetics interact directly with the C-terminus, which is thought to reside intracellularly, this portion of the channel protein clearly influences the actions of both anesthetics.

Anesthetics, Dissociative↗
PubMed1995

Key residues for membrane binding, oligomerization, and pore forming activity of staphylococcal alpha-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification.

The alpha-hemolysin (alpha HL) polypeptide is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into lipid bilayers to form cylindrical heptameric pores 1-2 nm in effective internal diameter. We have individually replaced each charged residue (79 of 293 amino acids) and four neutral residues in alpha HL with cysteine, which is not found in the wild-type protein. The properties of these mutants have been examined before and after modification with the 450-Da dianionic sulfhydryl reagent 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonate (IASD). This modification was highly informative as 28 of 83 modified polypeptides showed substantially reduced pore forming activity on rabbit erythrocytes (rRBC), while only five of the unmodified cysteine mutants were markedly affected. Through detailed examination of the phenotypes of the mutant and modified hemolysins, we have pinpointed residues and regions in the alpha HL polypeptide chain that are important for binding to rRBC, oligomer formation and pore activity. Residues in both the N-terminal (Arg-66 and Glu-70) and C-terminal (Arg-200, Asp-254, Asp-255, and Asp-276) thirds of the protein are implicated in binding to cells. The His-35 replacement mutant modified with IASD was the only polypeptide in this study that failed to form SDS-resistant oligomers on rRBC. Altered hemolysins that formed oligomers but failed to lyse rRBC represented the most common defect. These alterations were clustered in the central glycine-rich loop, which has previously been implicated as a component of the lumen of the membrane-spanning channel, and in the regions flanking the loop. Alterations in mutant and modified hemolysins with the same defect were also scattered between the N terminus and His-48, in keeping with previous suggestions that an N-terminal segment and the central loop cooperate in the final step of pore assembly.

Amino Acid Sequence↗
PubMed1995

Interactions between residues in staphylococcal alpha-hemolysin revealed by reversion mutagenesis.

alpha-Hemolysin (alpha HL), a pore-forming polypeptide of 293 amino acids, is secreted by Staphylococcus aureus as a water-soluble monomer. Residues that play key roles in the formation of functional heptameric pores on rabbit red blood cells (rRBC) have been identified previously by site-directed mutagenesis. alpha HL-H35N, in which the histidine at position 35 of the wild-type sequence is replaced with asparagine, is nonlytic and is arrested in assembly as a heptameric prepore. In this study, second-site revertants of H35N that have the ability to lyse rRBC were generated by error-prone PCR under conditions designed to produce single base changes. The analysis of 22 revertants revealed new codons clustered predominantly in three distinct regions of the H35N gene. One cluster includes amino acids 107-111 (four revertants) and another residues 144-155 (five revertants). These two clusters flank the central glycine-rich loop of alpha HL, which previously has been implicated in formation of the transmembrane channel, and encompass residues Lys-110 and Asp-152 that, like His-35, are crucial for lytic activity. The third cluster lies in the region spanning amino acids 217-228 (eight revertants), a region previously unexplored by mutagenesis. Single revertants were found at amino acid positions 84 and 169. When compared with H35N, the heptameric prepores formed by the revertants underwent more rapid conversion to fully assembled pores, as determined by conformational analysis by limited proteolysis. The rate of conversion to the fully assembled pore was strongly correlated with hemolytic activity. Previous work has suggested that the N terminus of alpha HL and the central loop cooperate in the final step of assembly. The present study suggests that the key N-terminal residue His-35 operates in conjunction with residues flanking the loop and C-terminal residues in the region 217-228. Hence, reversion mutagenesis extends the linear analysis that has been provided by direct point mutagenesis.

Amino Acid Sequence↗
PubMed1995

A photogenerated pore-forming protein.

BACKGROUND: The permeabilization of cells with bacterial pore-forming proteins is an important technique in cell biology that allows the exchange of small reagents into the cytoplasm of a cell. Another notable technology is the use of caged molecules whose activities are blocked by addition of photoremovable protecting groups. This allows the photogeneration of reagents on or in cells with spatial and temporal control. Here, we combine these approaches to produce a caged pore-forming protein for the controlled permeabilization of cells. RESULTS: 2-Bromo-2-(2-nitrophenyl)acetic acid (BNPA), a water-soluble cysteine-directed reagent for caging peptides and proteins with the alpha-carboxy-2-nitrobenzyl (CNB) protecting group, was synthesized. Glutathione (gamma-Glu-Cys-Gly) was released in high yield from gamma-Glu-CysCNB-Gly by irradiation at 300 nm. Based on this finding, scanning mutagenesis was used to find a single-cysteine mutant of the pore-forming protein staphylococcal alpha-hemolysin (alpha HL) suitable for caging. When alpha HL-R104C was derivatized with BNPA, pore-forming activity toward rabbit erythrocytes was lost. Near UV irradiation led to regeneration of the cysteine sulfhydryl group and the restoration of pore-forming activity. CONCLUSIONS: Caged pore-forming proteins are potentially useful for permeabilizing one cell in a collection of cells or one region of the plasma membrane of a single cell. Therefore, alpha HL-R104C-CNB and other caged proteins designed to create pores of various diameters should be useful for many purposes. For example, the ability to introduce reagents into one cell of a network or into one region of a single cell could be used in studies of neuronal modulation. Further, BNPA should be generally useful for caging cysteine-containing peptides and single-cysteine mutant proteins to study, for example, cell signaling or structural changes in proteins.

Animals↗
PubMed1995

An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch.

BACKGROUND: Studies of the mechanisms by which certain water-soluble proteins can assemble into lipid bilayers are relevant to several areas of biology, including the biosynthesis of membrane and secreted proteins, virus membrane fusion and the action of immune proteins such as complement and perforin. The alpha-hemolysin (alpha HL) protein, an exotoxin secreted by Staphylococcus aureus that forms heptameric pores in lipid bilayers, is a useful model for studying membrane protein assembly. In addition, modified alpha HL might be useful as a component of biosensors or in drug delivery. We have therefore used protein engineering to produce variants of alpha HL that contain molecular triggers and switches with which pore-forming activity can be modulated at will. Previously, we showed that the conductance of pores formed by the mutant hemolysin alpha HL-H5, which contains a Zn(II)-binding pentahistidine sequence, is blocked by Zn(II) from either side of the lipid bilayer, suggesting that residues from the pentahistidine sequence line the lumen of the transmembrane channel. RESULTS: Here we show that Zn(II) can arrest the assembly of alpha HL-H5 before pore formation by preventing an impermeable oligomeric prepore from proceeding to the fully assembled state. The prepore is a heptamer. Limited proteolysis shows that, unlike the functional pore, the prepore contains sites near the amino terminus of the polypeptide chain that are exposed to the aqueous phase. Upon removal of the bound Zn(II) with EDTA, pore formation is completed and the sites near the amino terminus become occluded. Conversion of the prepore to the active pore is the rate-determining step in assembly and cannot be reversed by the subsequent addition of excess Zn(II). CONCLUSIONS: The introduction of a simple Zn(II)-binding motif into a pore-forming protein has allowed the isolation of a defined intermediate in assembly. Genetically-engineered switches for trapping and releasing intermediates that are actuated by metal coordination or other chemistries might be generally useful for analyzing the assembly of membrane proteins and other supramolecular structures.

Bacterial Toxins↗
PubMed1995

Restoration of pore-forming activity in staphylococcal alpha-hemolysin by targeted covalent modification.

In previous studies, the replacement of His35 in the pore-forming protein alpha-hemolysin (alpha HL) with Leu, Ile, Pro, Arg, Ser, Thr or Cys yielded inactive polypeptides. Here, we show that modification of the inactive single-cysteine mutant alpha HL-H35C with iodoacetamide, to form H35CamC, generates significant pore-forming activity. The closely related polypeptides H35N and H35Q have, respectively, essentially no activity and greatly reduced activity. The modified residue in H35CamC, S-carboxamidomethyl-cysteine, mimicks histidine in volume, polarity and hydrogen bonding potential, but is unable to ionize. Unmodified H35C is defective in the final step of pore formation: the conversion of an inactive heptameric membrane-bound assembly intermediate to a structure containing open channels. It is this step in assembly that is ameliorated in H35CamC.

Animals↗
PubMed1994

Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore.

Elucidation of the accurate subunit stoichiometry of oligomeric membrane proteins is fraught with complexities. The interpretations of chemical cross-linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staphylococcal alpha-hemolysin (alpha HL), a homooligomeric toxin that forms channels in cell membranes, was believed to possess six subunits arranged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indicate that the alpha HL oligomer is a heptamer. Self-rotation functions calculated using x-ray diffraction data from single crystals of alpha HL oligomers show a sevenfold axis of rotational symmetry. The alpha HL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of alpha HL heteromers formed from subunits with the charge of wild-type alpha HL and subunits with additional negative charge generated by targeted chemical modification of a single-cysteine mutant. These data establish the heptameric oligomerization state of the alpha HL transmembrane pore both in three-dimensional crystals and on a biological membrane.

Animals↗
PubMed1994

Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal alpha-hemolysin.

Structural changes in staphylococcal alpha-hemolysin (alpha HL) that occur during oligomerization and pore formation on membranes have been examined by using a simple gel-shift assay to determine the rate of modification of key single-cysteine mutants with the hydrophilic sulfhydryl reagent, 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonate (IASD). The central glycine-rich loop of alpha HL lines the lumen of the transmembrane channel. A residue in the loop remains accessible to IASD after assembly, in keeping with the ability of the pore to pass molecules of approximately 1000 Da. By contrast, residues near the N-terminus, which are critical for pore function, become deeply buried during oligomerization, while a residue at the extreme C-terminus increases in reactivity after assembly, consistent with a location in the part of the pore that projects from the surface of the lipid bilayer.

Animals↗
PubMed1994

Differential phosphorylation of neuronal substrates by catalytic subunits of Aplysia cAMP-dependent protein kinase with alternative N termini.

cAMP-dependent protein kinase (PKA) is an important participant in neuronal modulation: the ability of neurons to change their properties in response to external stimuli. In Aplysia mechanosensory neurons, PKA plays roles in both short and long term presynaptic facilitation, which is a simple model for learning and memory. PKA in Aplysia is a collection of structurally and functionally diverse regulatory and catalytic (C) subunits. We have argued that this diversity may in part account for the ability of the enzyme to take part in neuronal events that are spatially and temporally separated. Here, we add credence to this hypothesis by showing that C subunits of Aplysia PKA with alternative N termini target different substrates in subcellular fractions from Aplysia neurons, despite their similar actions on synthetic peptide substrates. Purified recombinant CAPL-AN1A1, which has an N terminus that is homologous to the myristylated sequence described in mammals, catalyzes the formation of two phosphoproteins of 24 and 8 kDa more rapidly than CAPL-AN2A1, which has a distinct N terminus weakly related to that of the yeast TPK1 gene product. The 24-kDa phospoprotein, but not the 8-kDa species, is detected in taxol-stabilized microtubules, suggesting that it is associated with the cytoskeleton. CAPL-AN2A1, in contrast, generates a 55-kDa phosphoprotein that is not observed with CAPL-AN1A1. The 55-kDa species is found in the detergent supernatant of the cytoskeleton fraction. Differential targeting of substrates by C subunits of PKA may therefore contribute to the ability of this kinase to play multiple roles in neuronal modulation.

Animals↗