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1.
Chem Commun (Camb) ; 54(13): 1591-1594, 2018 Feb 08.
Article in English | MEDLINE | ID: mdl-29368774

ABSTRACT

Photopharmaceuticals can, in principle, be created by linking photoswitchable moieties to bioactive molecules. However, a general strategy for converting a therapeutic agent into its photoswitchable version is not currently available. Herein we propose a generalizable, modular approach for obtaining light controllable bioactive agents by modifying the scaffold of a protein affinity reagent using an azobenzene photoswitch.


Subject(s)
Peptide Fragments/chemistry , Photoaffinity Labels/chemistry , Proto-Oncogene Proteins c-fyn/chemistry , Azo Compounds/chemistry , Azo Compounds/radiation effects , Chymases/antagonists & inhibitors , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/radiation effects , Humans , Peptide Fragments/radiation effects , Photoaffinity Labels/radiation effects , Protein Folding/drug effects , Proto-Oncogene Proteins c-fyn/radiation effects , Sulfanilic Acids/chemistry , Sulfanilic Acids/radiation effects , Ultraviolet Rays
2.
Chem Commun (Camb) ; 51(65): 12981-4, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-26176021

ABSTRACT

Azonium ions formed by p-amino substituted azo compounds with both ortho- and meta-methoxy substituents exhibit strong absorbance in far-red and near infrared spectral region. The compounds undergo robust photoswitching in aqueous solution and exhibit a range of thermal relaxation rates from 10 µs-100 ms.


Subject(s)
Azo Compounds/chemistry , Infrared Rays , Ions/chemistry , Isomerism , Light , Photochemical Processes
3.
Photochem Photobiol Sci ; 14(9): 1729-36, 2015 Sep 26.
Article in English | MEDLINE | ID: mdl-26204102

ABSTRACT

A photo-controlled version of the engrailed homeodomain (zENG) was created by inserting the homeodomain into a surface loop of a circularly permuted version of the photoactive yellow protein (cPYP). The two proteins fold independently as judged by NMR and fluorescence denaturation measurements. In the dark, the affinity of the zENG domain for its cognate DNA is inhibited >100-fold compared to wild-type zENG. Blue-light irradiation of the hybrid protein leads to enhanced conformational dynamics of the cPYP portion and a two-fold enhancement of the DNA binding affinity of the zENG domain. These results suggest that insertion into a surface loop of cPYP can be a general approach for conferring an initial level of photo-control on a given target protein. Focussed mutation/selection strategies may then be used to enhance the degree of photo-control.


Subject(s)
Bacterial Proteins/chemistry , DNA/chemistry , Homeodomain Proteins/chemistry , Light , Luminescent Proteins/chemistry , Transcription Factors/chemistry , Bacterial Proteins/genetics , Chromatography, Liquid , Escherichia coli , Homeodomain Proteins/genetics , Isomerism , Luminescent Proteins/genetics , Models, Molecular , Photochemical Processes , Protein Conformation , Protein Denaturation , Spectrum Analysis , Transcription Factors/genetics
4.
Chem Commun (Camb) ; 50(93): 14613-5, 2014 Dec 04.
Article in English | MEDLINE | ID: mdl-25311049

ABSTRACT

A tetra-ortho-chloro substituted azobenzene unit was incorporated into a photoswitchable tethered ligand for ionotropic glutamate receptors. This compound confers the modified protein with the unusual optical responses of the substituted azo scaffold permitting channel opening with yellow and red light and channel closing with blue light.


Subject(s)
Azo Compounds/chemistry , Ion Channels/metabolism , Ion Channels/radiation effects , Receptors, Glutamate/metabolism , Receptors, Glutamate/radiation effects , HEK293 Cells , Humans , Ion Channels/chemistry , Ligands , Molecular Structure , Optical Phenomena , Receptors, Glutamate/chemistry
5.
Biophys J ; 84(1): 612-22, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12524314

ABSTRACT

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10(-8) cm(2)s(-1). Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10(-8) cm(2)s(-1). These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.


Subject(s)
Fluorescence Resonance Energy Transfer/methods , Gramicidin/chemistry , Lipid Bilayers/chemistry , Microscopy, Video/methods , Patch-Clamp Techniques/methods , Carbocyanines , Electrochemistry/methods , Feasibility Studies , Ion Channels/chemistry , Liposomes/chemistry , Membrane Potentials , Membranes, Artificial , Motion , Protein Conformation
6.
Bioconjug Chem ; 12(4): 594-602, 2001.
Article in English | MEDLINE | ID: mdl-11459465

ABSTRACT

Single-molecule spectroscopies in combination with single-channel patch-clamp measurements have the potential for providing new information on ion channel gating processes. Fluorescent gramicidin derivatives could provide a means for calibrating such experiments since the structure of the open channel is known and the mechanism of gating (peptide dimerization) is generally agreed. We describe here the synthesis and characterization of two pairs of gramicidin derivatives that should prove useful for such studies. They contain robust fluorophores, undergo resonance energy transfer (FRET) when they dimerize, and have single-channel properties close to those of the wild-type channel.


Subject(s)
Fluorescent Dyes/chemical synthesis , Gramicidin/analogs & derivatives , Ion Channel Gating , Membrane Lipids/chemistry , Peptides/chemistry , Dimerization , Ion Channels/chemistry , Patch-Clamp Techniques , Spectrometry, Fluorescence
7.
Protein Eng ; 14(12): 983-91, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11809929

ABSTRACT

Incorporation of the photoisomerizable amino acid phenylazophenylalanine (PAP) into enzyme structures has been proposed as a strategy for photoswitching enzyme activity. To evaluate the strengths and limitations of this approach to enzyme photo-control, we performed a kinetic analysis of RNase S analogues containing PAP in positions 4, 7, 8, 10, 11 or 13. For an enzyme containing a single PAP group, the maximum extent of photoconversion (between approximately 96% trans/4% cis and 10% trans/90% cis under standard conditions) sets a limit on the maximum fold change in the initial rate of approximately 25-fold, if the cis form is the more active isomer, and approximately 10-fold if the trans form is more active. This extent of photoswitching was not realized in the present case because the effects of photoisomerization on kinetic constants were small and distributed among effects on S-peptide binding, substrate binding and the rate of the chemical step. These results suggest that photoisomerization could substantially alter enzyme kinetic constants but that a directed combinatorial approach might be required for realizing maximal photo-control in such systems. The limit set by the extent of photoconversion might be overcome by coupling multiple PAP groups to one enzyme or by altering the behaviour of a system that required oligomerization for activity.


Subject(s)
Phenylalanine/analogs & derivatives , Phenylalanine/chemistry , Ribonucleases/chemistry , Amino Acid Sequence , Azo Compounds/chemistry , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Isomerism , Kinetics , Molecular Sequence Data , Mutation , Photochemistry , Ribonucleases/genetics , Ribonucleases/metabolism
8.
Proc Natl Acad Sci U S A ; 97(8): 3803-8, 2000 Apr 11.
Article in English | MEDLINE | ID: mdl-10760254

ABSTRACT

The alpha-helix is a key structural element in a wide range of peptides and proteins. We report here the design, synthesis, and characterization of a modified peptide in which the helix content can be reversibly photoregulated. The peptide contains two cysteine residues that are cross-linked by an azobenzene derivative in an intramolecular fashion. In accordance with the design, the photoisomerization of the azobenzene cross-linker from the trans to the cis form causes a large increase in the helix content of the peptide, in water.


Subject(s)
Peptides/chemistry , Amino Acid Sequence , Circular Dichroism , Isomerism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Spectrophotometry, Ultraviolet , Temperature
9.
Biophys J ; 78(3): 1335-48, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10692320

ABSTRACT

A dimeric alamethicin analog with lysine at position 18 in the sequence (alm-K18) was previously shown to form stable anion-selective channels in membranes at pH 7.0 [Starostin, A. V., R. Butan, V. Borisenko, D. A. James, H. Wenschuh, M. S. Sansom, and G. A. Woolley. 1999. Biochemistry. 38:6144-6150]. To probe the charge state of the conducting channel and how this might influence cation versus anion selectivity, we performed a series of single-channel selectivity measurements at different pH values. At pH 7.0 and below, only anion-selective channels were found with P(K(+))/P(Cl(-)) = 0. 25. From pH 8-10, a mixture of anion-selective, non-selective, and cation-selective channels was found. At pH > 11 only cation-selective channels were found with P(K(+))/P(Cl(-)) = 4. In contrast, native alamethicin-Q18 channels (with Gln in place of Lys at position 18) were cation-selective (P(K(+))/P(Cl(-)) = 4) at all pH values. Continuum electrostatics calculations were then carried out using an octameric model of the alm-K18 channel embedded in a low dielectric slab to simulate a membrane. Although the calculations can account for the apparent pK(a) of the channel, they fail to correctly predict the degree of selectivity. Although a switch from cation- to anion-selectivity as the channel becomes protonated is indicated, the degree of anion-selectivity is severely overestimated, suggesting that the continuum approach does not adequately represent some aspect of the electrostatics of permeation in these channels. Side-chain conformational changes upon protonation, conformational changes, and deprotonation caused by permeating cations and counterion binding by lysine residues upon protonation are considered as possible sources of the overestimation.


Subject(s)
Alamethicin/analogs & derivatives , Alamethicin/chemistry , Ion Channels/chemistry , Ion Channels/physiology , Lysine , Dimerization , Hydrogen-Ion Concentration , Kinetics , Membrane Potentials , Models, Biological , Patch-Clamp Techniques , Polystyrenes , Static Electricity , Surface Properties
10.
Biochem Biophys Res Commun ; 267(1): 394-7, 2000 Jan 07.
Article in English | MEDLINE | ID: mdl-10623630

ABSTRACT

A mutant of Photinus pyralis luciferase in which all four native cysteine residues are converted to serines retains about 10% of wild-type activity. This mutant should prove useful as a starting point for the introduction of biophysical probes of conformational changes associated with enzyme function. The activities of the cysteine-free mutant and others in which two or three cysteines are converted to serines suggest, however, that small chemical changes can have substantial and interdependent effects on bioluminescence. The introduction of probes should therefore be approached cautiously.


Subject(s)
Coleoptera/enzymology , Cysteine , Luciferases/chemistry , Luciferases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Luciferases/genetics , Luminescent Measurements , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Serine
11.
Novartis Found Symp ; 225: 62-9; discussion 69-73, 1999.
Article in English | MEDLINE | ID: mdl-10472048

ABSTRACT

The peptide alamethicin provides a system for engineering ion channel charge selectivity. To define alamethicin charge selectivity experimentally, we measured single-channel current-voltage relationships in KCl gradients using covalently linked peptide dimers. Two factors were found to contribute to the charge selectivity of these channels: (i) the ionic strength of the surrounding solutions; and (ii) the distribution of fixed charge on the peptide. Native alamethicin channels exhibited either cation selectivity or anion selectivity depending on which end of the channel was at the low salt side of the membrane. When the glutamine residue at position 18 in the sequence was replaced with a lysine residue, an anion-selective channel was obtained regardless of which end of the channel was at the low salt side of the membrane.


Subject(s)
Alamethicin/chemistry , Anti-Bacterial Agents/chemistry , Protein Engineering , Amino Acid Sequence , Drug Design , Electrochemistry , Molecular Sequence Data
12.
J Pept Res ; 53(5): 560-8, 1999 May.
Article in English | MEDLINE | ID: mdl-10424352

ABSTRACT

The synthesis of a conformationally constrained photoisomerizable amino acid, phenylazo-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid (PATIC), is described. This amino acid can be incorporated into peptides using standard Fmoc procedures and can be accommodated within alpha-helical structures albeit with some loss of stability of the structure. PATIC can serve as a useful building block for the synthesis of photoregulated peptides and proteins.


Subject(s)
Azo Compounds/chemical synthesis , Isoquinolines/chemical synthesis , Tetrahydroisoquinolines , Azo Compounds/chemistry , Azo Compounds/radiation effects , Circular Dichroism , Isomerism , Isoquinolines/chemistry , Isoquinolines/radiation effects , Magnetic Resonance Spectroscopy , Models, Molecular , Photochemistry , Protein Conformation , Spectrophotometry, Ultraviolet
13.
Biochemistry ; 38(19): 6144-50, 1999 May 11.
Article in English | MEDLINE | ID: mdl-10320341

ABSTRACT

The peptide alamethicin self-assembles to form helix bundle ion channels in membranes. Previous macroscopic measurements have shown that these channels are mildly cation-selective. Models indicate that a source of cation selectivity is a zone of partial negative charge toward the C-terminal end of the peptide. We synthesized an alamethicin derivative with a lysine in this zone (replacing the glutamine at position 18 in the sequence). Microscopic (single-channel) measurements demonstrate that dimeric alamethicin-lysine18 (alm-K18) forms mildly anion-selective channels under conditions where channels formed by the parent peptide are cation-selective. Long-range electrostatic interactions can explain the inversion of ion selectivity and the conductance properties of alamethicin channels.


Subject(s)
Alamethicin/chemistry , Ion Channels/metabolism , Alamethicin/chemical synthesis , Amino Acid Sequence , Anions/metabolism , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Fees and Charges , Ion Channels/chemical synthesis , Molecular Sequence Data , Peptide Biosynthesis , Potassium Chloride/metabolism , Sequence Homology, Amino Acid
14.
Anal Biochem ; 264(1): 26-33, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9784184

ABSTRACT

A sensitive assay for ribonuclease A activity based on the relief of fluorescence quenching within a defined oligomeric substrate (5' fluorescein-AAAArUAAAA-3'-rhodamine) is described. The substrate can be produced using an automated nucleic acid synthesizer and commercially available reagents. Together with a nonfluorescent cosubstrate (5'-dimethoxytrityl-AAAArUAAAA), the compound can be used to determine kinetic constants for the first step (transphosphorylation) of the ribonuclease-catalyzed reaction. These measurements should be useful for structure-based analyses of ribonuclease activity since a crystal structure has been determined for a closely analogous enzyme-inhibitor complex.


Subject(s)
Ribonuclease, Pancreatic/analysis , Fluoresceins , Fluorescent Dyes/metabolism , Kinetics , Oligonucleotide Probes/metabolism , Oligonucleotides , Rhodamines , Ribonuclease, Pancreatic/metabolism , Substrate Specificity
15.
Structure ; 6(6): 783-92, 1998 Jun 15.
Article in English | MEDLINE | ID: mdl-9655831

ABSTRACT

BACKGROUND: Antiamoebin is a member of the peptaibol family of polypeptides and has a unique antibiotic activity: it acts as an antiamoebic agent, but does not effectively haemolyze erythrocytes even though it does exhibit membrane-modifying activity. RESULTS: The structure of antiamoebin I has been determined by X-ray crystallography at 1.4 A resolution. The molecule forms a helical structure, which, as a result of the presence of a number of proline and hydroxyproline residues, has a deep bend in the middle. Circular dichroism spectroscopy, single-channel conductance studies and fluorescence diffusion studies suggest a mode of ion transport that is entirely different from that of the other two members of the peptaibol family (alamethicin and zervamicin) whose structures and functions have been examined in detail. CONCLUSIONS: The structure of the polypeptide has been determined and a functional model for its mode of action in membranes is presented. Although under some conditions antiamoebin may form ion channels, unlike the closely related alamethicin and zervamicin polypeptides, its major membrane-modifying activity appears to be as an ion carrier.


Subject(s)
Amebicides/chemistry , Anti-Bacterial Agents/chemistry , Carrier Proteins/chemistry , Peptides , Alamethicin/chemistry , Amebicides/pharmacology , Anti-Bacterial Agents/pharmacology , Carrier Proteins/pharmacology , Circular Dichroism , Crystallography, X-Ray , Membranes/drug effects , Models, Chemical , Models, Molecular , Peptaibols , Proline
16.
Biochemistry ; 36(45): 13873-81, 1997 Nov 11.
Article in English | MEDLINE | ID: mdl-9374865

ABSTRACT

Alamethicin channels are prototypical helix bundles that may serve as tractable models for more complex protein ion channels. Solid-phase peptide synthesis of alamethicin analogues using FMOC-amino acid fluorides followed by chemical dimerization of these peptides facilitates structure-function studies of particular channel states in bilayer membranes. State 3 in particular, tentatively assigned to a hexameric helix bundle, is sufficiently long-lived that current-voltage measurements can be made during the lifetime of an individual channel opening. Molecular models of hexameric helix bundles, generated using restrained molecular dynamics with simulated annealing, indicate that a Gln7-->Asn7 (Q7-->N7) mutation will increase channel diameter locally. Experimentally, the conductance of state 3 of the N7-alm channel is found to be larger than that of the Q7-alm channel when ion flow is in the usual direction (cations entering the C-terminal end of the channel). When ion flow is in the opposite direction, no difference in the conductances of state 3 of Q7 and state 3 of N7 channels is observed. These results indicate that the effect of a change in pore diameter at position 7 is dependent on the magnitude of other barriers to permeation and that these barriers are voltage-dependent.


Subject(s)
Alamethicin/chemical synthesis , Asparagine/genetics , Glutamine/genetics , Ion Channels/chemical synthesis , Protein Structure, Secondary , Alamethicin/chemistry , Amino Acid Sequence , Amino Acid Substitution/genetics , Dimerization , Electric Conductivity , Ion Channels/genetics , Ion Channels/physiology , Membrane Potentials , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptides/chemical synthesis , Structure-Activity Relationship
17.
Biophys J ; 73(5): 2465-75, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9370440

ABSTRACT

The channel-forming properties of two analogs of gramicidin, gramicidin-ethylenediamine (gram-EDA), and gramicidin-N,N-dimethylethylenediamine (gram-DMEDA) were studied in planar lipid bilayers, using protons as the permeant ion. These peptides have positively charged amino groups tethered to their C-terminal ends via a linker containing a carbamate group. Gram-DMEDA has two extra methyl groups attached to the terminal amino group, making it a bulkier derivative. The carbamate groups undergo thermal cis-trans isomerization on the 10-100-ms time scale. The conductance behavior of gram-EDA is found to be markedly voltage dependent, whereas the behavior of gram-DMEDA is not. In addition, voltage affects the cis-trans ratios of the carbamate groups of gram-EDA, but not those of gram-DMEDA. A model is proposed to account for these observations, in which voltage can promote the binding of the terminal amino group of gram-EDA to the pore in a "ball-and-chain" fashion. The bulkiness of the gram-DMEDA derivative prevents this binding.


Subject(s)
Gramicidin/chemistry , Ion Channels/chemistry , Amino Acid Sequence , Carbamates/chemistry , Carbamates/metabolism , Chlorides/metabolism , Electric Conductivity , Electrophysiology , Gramicidin/metabolism , Hydrogen-Ion Concentration , Ion Channel Gating , Ion Channels/metabolism , Isomerism , Lipid Bilayers/metabolism , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Protons
18.
Biophys J ; 73(2): 770-8, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9251793

ABSTRACT

Covalent dimers of alamethicin form conducting structures with gating properties that permit measurement of current-voltage (I-V) relationships during the lifetime of a single channel. These I-V curves demonstrate that the alamethicin channel is a rectifier that passes current preferentially, with voltages of the same sign as that of the voltage that induced opening of the channel. The degree of rectification depends on the salt concentration; single-channel I-V relationships become almost linear in 3 M potassium chloride. These properties may be qualitatively understood by using Poisson-Nernst-Planck theory and a modeled structure of the alamethicin pore.


Subject(s)
Alamethicin/chemistry , Potassium Channels/chemistry , Potassium Channels/physiology , Protein Structure, Secondary , Amino Acid Sequence , Dimerization , Ion Channel Gating , Kinetics , Lipid Bilayers , Membrane Potentials , Models, Biological , Models, Structural , Molecular Sequence Data , Phosphatidylcholines , Potassium Chloride , Static Electricity , Thermodynamics
19.
Biochemistry ; 36(30): 9237-45, 1997 Jul 29.
Article in English | MEDLINE | ID: mdl-9230057

ABSTRACT

We studied the mechanism of membrane permeabilization by the 18L model peptide (GIKKFLGSIWKFIKAFVG), which features the consensus class L sequence averaged from the number of naturally occurring lytic peptides. Two aspects of membrane lipid composition significantly affected peptide-membrane interactions: the presence of acidic lipids and, in zwitterionic membranes, and the presence of nonbilayer forming lipids. In zwitterionic membranes, 18L peptide destabilizes the membrane, leading to a transient formation of large defects in the membrane which result generally in contents leakage, but in the presence of bilayer-bilayer contact can alternatively lead to vesicle fusion. In membranes containing acidic lipids (DOPC:DOPG, DOPG), 18L caused leakage but not fusion, probably due to mutual repulsion of acidic vesicles. While the extent of contents leakage was approximately the same as for zwitterionic membranes, the kinetics of leakage could be resolved only by using stopped-flow, leakage being essentially complete within the first minute. Previously, we reported that apolipoprotein (class A) and lytic (class L) peptide analogs have opposing effects on some properties of biological membranes. This reciprocal effect of 18L and Ac-18A-NH2, class A model peptide, is restricted to membranes with a high propensity for nonbilayer phase formation (DOPE, Me-DOPE, DOPC:DOPE, DOPC:Me-DOPE). The decrease in the content of nonbilayer phase forming lipid or the addition of acidic lipids reduces or eliminates the reciprocal effects. This suggests the importance of nonbilayer phase propensity for certain functions of biological membranes.


Subject(s)
Membrane Lipids/metabolism , Membrane Lipids/physiology , Peptide Fragments/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Ion Channels/chemistry , Liposomes , Membrane Fusion/drug effects , Molecular Sequence Data , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Permeability/drug effects , Protein Binding
20.
Biochemistry ; 36(6): 1343-8, 1997 Feb 11.
Article in English | MEDLINE | ID: mdl-9063882

ABSTRACT

We have synthesized a novel thiol reagent, 2-[(methylsulfonyl)thio]ethyl [N-(N,N-dimethylamino)ethyl]carbamate (MTSAC), that contains a carbamate functional group as well as a (positively charged) terminal amino group. The carbamate C-N bond isomerizes on a millisecond time scale and significantly alters the three-dimensional shape of the reagent. The behavior of this reagent was contrasted with that of the commonly used thiol reagent, [(methylsulfonyl)thio]ethylamine MTSEA [Akabas, M. H., & Karlin, A. (1995) Biochemistry 34, 12496-12500], with respect to its effect on single-channel currents passing through modified gramicidin channels. While both reagents decreased single-channel currents, the MTSAC-treated channels also showed a pattern of steps in the current recordings on the time scale of the carbamate bond isomerization. Moreover, the pattern and size of these steps were sensitive to the location of the thiol-reactive site in relation to the channel entrance. Thus, MTSAC may prove useful as a reagent for establishing the proximity to the pore in studies of ion channel proteins of unknown structure.


Subject(s)
Carbamates/chemical synthesis , Ion Channels/chemistry , Models, Molecular , Sulfones/chemical synthesis , Anti-Bacterial Agents/chemistry , Gramicidin/analogs & derivatives , Gramicidin/chemistry , Gramicidin/metabolism , Ion Channels/metabolism , Kinetics , Spectrophotometry, Ultraviolet , Sulfhydryl Reagents
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