About us. Fact Sheet. Objective Electrochemical investigations of biological processes have provided a wealth of information on the structure-function relationship of redox enzymes, while the underlying technology has formed the basis for biosensors such as the extremely successful glucose biosensor.
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- Membrane Electrochemistry.
While electrochemistry of globular redox-enzymes is limited by poor control of the surface-protein interface, the absence of control with membrane proteins has made it impossible to study them electrochemically. Donnan potentials, created across the vesicular bilayer membrane by polyelectrolytes, are used to induce the conformational changes in alamethicin. The induced change of CD by the electric field across the membrane was not symmetrical with respect to the field direction. Tangential electric fields induce lateral movements of membrane components. The average apparent electric mobility, determined from the time course of the increase of EPL on the enriched hemisphere and of the decrease of EPL on the depleted hemisphere, was of the order of 3.
The distribution of PSI reaches a steady state when the diffusional, electrostatic, and other counteracting forces balance the electrophoretic driving force. A summary is given of investigations that show that lipid-water mixtures near lamellar-nonlamellar phase boundaries are under an elastic curvature stress that may couple to conformational changes of imbedded membrane proteins. Lamellar-nonlamellar phase transitions are shown to be the result of geometrically frustrated competing free energies associated with a spontaneous tendency for lipid layers to bend and with the hydrocarbon packing configurations in the core of the lipid layers.
Experimental procedures for measuring the magnitude of the curvature energy and for altering the competition between curvature and packing are described. Mechanisms whereby frustrated bilayer curvature elasticity may couple to membrane protein conformations are outlined, and experiments that indicate a correlation between elastic curvature stress and protein function are summarized.
Dye indicators of membrane potential have been available for the past 20 years and have been employed in numerous studies of cell physiology. Fast dyes include compounds with styryl, oxonol, and merocyanine chromophores that are engineered to stain the plasma membrane.
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Generally fast dyes have small sensitivities to voltage changes but are very useful for detecting voltage transients or mapping voltage differences along the surface of a cell. Slow dyes have delocalized charges, as in the cyanine or rhodamine chromophores, and usually operate via a potential-dependent redistribution between the extracellular medium and the cytosol. Techniques based on these indicators can be used to monitor both spatial and temporal variations in membrane potential with resolutions not possible with the more traditional electrode-based methodologies.
The contribution of hydration to the energetics of molecular conformation and assembly has been recognized for a long time, but has been difficult to measure. We use osmotic stress to measure the forces and energies in molecular assemblies. The same strategy can be used to measure the contribution of water to the chemical free energy change of individually functioning molecules. We describe, as examples, the contribution of water to the gating of membrane channels to the binding of oxygen to hemoglobin; and to the forces between bilayer membranes, within nonbilayer lipid assemblies, and on macromolecular surfaces.
From the magnitude of their energies we conclude that hydration-dehydration reactions play an important but neglected role in molecular function. Connexin protein, which forms gated channels through closely apposed cell membranes "gap junction channels" , also forms channels in single membranes.
The mechanisms by which single- and double-membrane connexin channels are formed, gated, and regulated are of biophysical interest, yet are largely unknown. Biophysical studies have been hindered by inaccessibility of cellular connexin channels and by the unique problems of connexin reconstitution. Connexin from isolated gap junctions and from monoclonal immunoaffinity purification from plasma membrane forms large, dynamically gated channels in liposomes and planar bilayers. These connexin channels may be single hemichannels—the subunits that span a single membrane in situ—and are accessible for detailed biophysical study.
Implications and possibilities for future studies of permeation, gating, and modulation are discussed. The structure of the compound eye of dipteran insects provides a unique preparation for investigation of functional coupling by gap junctions in photoreceptor axon terminals in an intact animal. Axon terminals of individual photoreceptors converge and synapse onto a pair of cells that form a visual element.
These terminals are electrically coupled by gap junctions. Characterization of the nature of the photoreceptor input at this synapse is a prerequisite to understanding how the stimulus is encoded by a visual element. Control of coupling is imperative to isolate individual photoreceptor cells and preserve their optical disparity. An anatomical substrate that possibly underlies hyperacuity in this animal is shown by optically staining the photoreceptor mosaic in the retina. Although photoreceptor axon terminals are electrically coupled, their gap junctions do not allow dye coupling of the cells.
The hypothesis that the gap junctions are voltage sensitive was tested by adaptation of the animal to light or darkness and by injection of depolarizing and hyperpolarizing current directly into the recorded axon terminal after dye injection. Control of the gap junctions by calcium concentration or pH in the terminal was tested by injection of buffers into the terminal. Ongoing experiments will better define these differences and dissect the control mechanism of electrical coupling of photoreceptor axon terminals in a complex but highly structured region of the nervous system.
The mitochondrial outer membrane contains a kDa protein called voltage-dependent anion-selective channel VDAC that forms channels with 3-nm pores through which metabolites travel between the cytoplasm and the mitochondrial spaces. Electron micrographs of two-dimensional crystals of these channels after freeze-drying, shadowing, and computer processing reveal detailed surface images of the channels. Both surfaces look very similar: most of the protein appears to be embedded in the membrane.
The ion selectivity of the channel and changes in it induced by site-directed mutations fit quite well to the fixed-charge theory of Teorell. The voltage-dependent closure of VDAC at both positive and negative potentials can be modulated by polyanions, osmotic pressure, aluminum hydroxide, and a soluble mitochondrial protein called the VDAC modulator. VDAC acts as a binding site for proteins perhaps through a domain located on the membrane surface. The increased interest in peptide ion channels as model systems for larger, physiological channels coincides with improvements in experimental techniques that make peptides more accessible and enable the determination of their structures.
This chapter surveys peptides from natural sources of less than 50 residues that are known to function as ion channels and for which three-dimensional structural information is available. Although the helix was initially recognized as a structural motif that made insertion of a peptide into the lipid bilayer energetically feasible, this survey suggests it is not unique.
While the magainins, the peptaibols, and the cecropins are examples of helical structures, the role of beta-sheet structures is apparent in tachyplesin and the defensins. Other peptides, such as the lantibiotics, are as yet structurally unclassifiable. A number of structural themes emerge from this analysis that help to explain how such small chemical entities can form pores that enable the transit of ions across the lipid bilayer.
Mutations in PMA1 , selected by growth resistance to hygromycin B, cause a depolarization of cellular membrane potential. Second-site mutations that partially revert the pma phenotype were identified within the membrane sector and provide strong evidence for coupling between cytoplasmic and membrane domains. However, a special emphasis is placed on the role of transmembrane segments 1 and 2, which provide the starting point for a more inclusive structural model of the membrane sector.
Proteins that emulate pore properties of the dihydropyridine-sensitive calcium channel or the nicotinic acetylcholine receptor have been designed and synthesized. Molecular models suggest that such structures satisfy geometric and functional requirements to constitute the pore-forming element of channel proteins. The designed proteins are synthesized by solid-phase methods and purified.
The single-channel conductance properties of designed proteins are studied in lipid bilayers. The synthetic proteins mimic the ionic conductance, selectivity, and pharmacological properties of authentic channel proteins. Synthetic proteins that represent segments of the authentic proteins that are not predicted to line an aqueous pore do not mimic the targeted biological activity.
The sequence of open and closed states measured from individual ion channels is fractal; namely, the pattern of openings and closings at one temporal resolution is similar to that viewed at other temporal resolutions.
This phenomenon suggests that ion channel proteins have 1 many states that are shallow energy minima, 2 different physical processes that cooperate to open and close the channel, 3 activation energy barriers between states that vary in time, and 4 a set of processes that have different widths in their distribution of activation energy barriers. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account.
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