Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 41
Filter
Add more filters










Publication year range
1.
Molecules ; 27(9)2022 Apr 20.
Article in English | MEDLINE | ID: mdl-35565996

ABSTRACT

Neuroactive amino acids derivatised at their carboxylate groups with a photolabile nitroindolinyl group are highly effective reagents for the sub-µs release of neuroactive amino acids in physiological solutions. However, the same does not apply in the case of calcium ion chelators. In this study, nitroindolinyl-caged BAPTA is found to be completely photostable, whereas nitroindolinyl-caged EDTA photolyses only when saturated with calcium ions.


Subject(s)
Calcium , Chelating Agents , Amino Acids , Calcium/metabolism , Calcium Chelating Agents , Ions , Photolysis
2.
Photochem Photobiol Sci ; 15(5): 604-8, 2016 05 11.
Article in English | MEDLINE | ID: mdl-27050155

ABSTRACT

The photolysis quantum yield, Qp, of 1-(2-nitrophenyl)ethyl phosphate (caged Pi) measured in the near-UV (342 nm peak with 60 nm half-bandwidth) is 0.53 and is based on results reported in 1978 (Biochemistry, 17, 1929-1935). This article amplifies methodology for determining that Qp in view of different recent estimates. Some general principles together with other examples relating to measurement of Qp values are discussed together with their relevance to biological research.


Subject(s)
Organophosphates/chemistry , Photolysis , Spectrophotometry, Ultraviolet , Ultraviolet Rays
4.
Phys Chem Chem Phys ; 15(6): 2177-83, 2013 Feb 14.
Article in English | MEDLINE | ID: mdl-23247608

ABSTRACT

The predicted structure has been calculated for a protein-based biosensor for inorganic phosphate (Pi), previously developed by some of us (Okoh et al., Biochemistry, 2006, 45, 14764). This is the phosphate binding protein from Escherichia coli labelled with two rhodamine fluorophores. Classical molecular dynamics and hybrid Car-Parrinello/molecular mechanics simulations allow us to provide molecular models of the biosensor both in the presence and in the absence of Pi. In the latter case, the rhodamine fluorophores maintain a stacked conformation in a 'face A to face B' orientation, which is different from the 'face A to face A' stacked orientation of free fluorophores in aqueous solution (Ilich et al., Spectrochim. Acta, Part A, 1996, 52, 1323). A protein conformation change upon binding Pi prevents significant stacking of the two rhodamines. In both states, the rhodamine fluorophores form hydrophobic contact with LEU291, without establishing significant hydrogen bonds with the protein. The accuracy of the models is established by a comparison between calculated and experimental absorption and circular dichroism spectra.


Subject(s)
Biosensing Techniques , Rhodamines/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Hydrophobic and Hydrophilic Interactions , Molecular Dynamics Simulation , Phosphate-Binding Proteins/chemistry , Phosphate-Binding Proteins/metabolism , Phosphates/chemistry , Protein Binding , Protein Structure, Tertiary , Rhodamines/chemistry
5.
Neuropharmacology ; 63(4): 624-34, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22609535

ABSTRACT

Photolysis is widely used in experimental neuroscience to isolate post-synaptic receptor activation from presynaptic processes, to determine receptor mechanisms in situ, for pharmacological dissection of signaling pathways, or for photostimulation/inhibition in neural networks. We have evaluated new caged neuroactive amino acids that use 4-methoxy-7-nitroindolinyl- (MNI) or 1-(2-nitrophenyl)ethoxycarbonyl (NPEC) photoprotecting groups to make caged ligands specific for glutamate receptor sub-types. Each was tested for interference with synaptic transmission and excitability and for receptor-specific actions in slice preparations. No adverse effects were found at glutamate receptors. At high concentration, MNI-caged, but not NPEC-caged ligands, interfered with GABA-ergic transmission. MNI-caged amino acids have sub-microsecond release times suitable for investigating mechanisms at fast synaptic receptors in situ. MNI-NMDA and MNI-kainate were synthesized and tested. MNI-NMDA showed stoichiometric release of chirally pure NMDA. Wide-field photolysis in cerebellar interneurons produced a fast-rising sustained activation of NMDA receptors, and localized laser photolysis gave a fast, transient response. Photolysis of MNI-kainate to release up to 4 µM kainate generated large inward currents at resting membrane potential in Purkinje neurons. Application of GYKI 53655 indicated that 40% of the current was due to AMPA receptor activation by kainate. Signaling via metabotropic glutamate receptors (mGluR) does not require fast release rates. NPEC cages are simpler to prepare but have slower photorelease. Photolysis of NPEC-ACPD or NPEC-DHPG in Purkinje neurons generated slow inward currents blocked by the mGluR type 1 antagonist CPCCOEt similar to the slow sEPSC seen with parallel fiber burst stimulation. NPEC-AMPA was also tested in Purkinje neurons and showed large sustained inward currents selective for AMPA receptors with little activation of kainate receptors. MNI-caged l-glutamate, NMDA and kainate inhibit GABA-A receptors with IC50 concentrations close to the maximum concentrations useful in receptor signaling experiments.


Subject(s)
Excitatory Amino Acid Agonists/pharmacology , Kainic Acid/analogs & derivatives , N-Methylaspartate/analogs & derivatives , Nerve Tissue Proteins/agonists , Receptors, Ionotropic Glutamate/agonists , Animals , Cerebellum/drug effects , Cerebellum/metabolism , Evoked Potentials/drug effects , Excitatory Amino Acid Agonists/adverse effects , Excitatory Amino Acid Agonists/radiation effects , Excitatory Amino Acid Antagonists/pharmacology , In Vitro Techniques , Indoles/chemistry , Interneurons/drug effects , Interneurons/metabolism , Kainic Acid/adverse effects , Kainic Acid/pharmacology , Kainic Acid/radiation effects , Ligands , N-Methylaspartate/adverse effects , N-Methylaspartate/pharmacology , N-Methylaspartate/radiation effects , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Nitro Compounds/chemistry , Photolysis , Protein Isoforms/agonists , Protein Isoforms/metabolism , Purkinje Cells/drug effects , Purkinje Cells/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Ionotropic Glutamate/antagonists & inhibitors , Receptors, Ionotropic Glutamate/metabolism , Synaptic Transmission/drug effects , Ultraviolet Rays
6.
Neuron ; 66(2): 235-47, 2010 Apr 29.
Article in English | MEDLINE | ID: mdl-20435000

ABSTRACT

Miniature synaptic currents have long been known to represent random transmitter release under resting conditions, but much remains to be learned about their nature and function in central synapses. In this work, we describe a new class of miniature currents ("preminis") that arise by the autocrine activation of axonal receptors following random vesicular release. Preminis are prominent in gabaergic synapses made by cerebellar interneurons during the development of the molecular layer. Unlike ordinary miniature postsynaptic currents in the same cells, premini frequencies are strongly enhanced by subthreshold depolarization, suggesting that the membrane depolarization they produce belongs to a feedback loop regulating neurotransmitter release. Thus, preminis could guide the formation of the interneuron network by enhancing neurotransmitter release at recently formed synaptic contacts.


Subject(s)
Interneurons/physiology , Neural Inhibition/physiology , Presynaptic Terminals/physiology , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolism , Animals , Cerebellum/metabolism , Cerebellum/physiology , Electrophysiology , Inhibitory Postsynaptic Potentials/physiology , Interneurons/metabolism , Microscopy, Electron , Miniature Postsynaptic Potentials/physiology , Presynaptic Terminals/metabolism , Rats , Rats, Sprague-Dawley , Synapses/metabolism , Synapses/physiology
7.
Proc Natl Acad Sci U S A ; 107(6): 2693-8, 2010 Feb 09.
Article in English | MEDLINE | ID: mdl-20133736

ABSTRACT

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins in the human genome. Events in the GPCR signaling cascade have been well characterized, but the receptor composition and its membrane distribution are still generally unknown. Although there is evidence that some members of the GPCR superfamily exist as constitutive dimers or higher oligomers, interpretation of the results has been disputed, and recent studies indicate that monomeric GPCRs may also be functional. Because there is controversy within the field, to address the issue we have used total internal reflection fluorescence microscopy (TIRFM) in living cells to visualize thousands of individual molecules of a model GPCR, the M(1) muscarinic acetylcholine receptor. By tracking the position of individual receptors over time, their mobility, clustering, and dimerization kinetics could be directly determined with a resolution of approximately 30 ms and approximately 20 nm. In isolated CHO cells, receptors are randomly distributed over the plasma membrane. At any given time, approximately 30% of the receptor molecules exist as dimers, and we found no evidence for higher oligomers. Two-color TIRFM established the dynamic nature of dimer formation with M(1) receptors undergoing interconversion between monomers and dimers on the timescale of seconds.


Subject(s)
Microscopy, Fluorescence/methods , Pirenzepine/analogs & derivatives , Receptor, Muscarinic M1/metabolism , Animals , Benzenesulfonates/chemistry , Binding, Competitive , CHO Cells , Carbocyanines/chemistry , Cell Membrane/metabolism , Cricetinae , Cricetulus , Fluorescent Dyes/chemistry , Humans , Kinetics , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Molecular Structure , Muscarinic Antagonists/chemistry , Muscarinic Antagonists/metabolism , Muscarinic Antagonists/pharmacology , Pirenzepine/metabolism , Pirenzepine/pharmacology , Protein Multimerization , Radioligand Assay , Receptor, Muscarinic M1/antagonists & inhibitors , Receptor, Muscarinic M1/genetics , Time Factors , Transfection
8.
Photochem Photobiol ; 85(5): 1089-96, 2009.
Article in English | MEDLINE | ID: mdl-19496988

ABSTRACT

Alpha-carboxy-4-nitrobenzyl phosphate 4 and its derived monomethyl phosphate ester 5 were synthesized and purified by anion-exchange chromatography. A gradient of LiCl was necessary for elution of the anion-exchange column to avoid unexpected thermal decarboxylation that occurred during vacuum evaporation when the volatile triethylammonium bicarbonate buffer was used. Photolysis of each compound was accompanied by decarboxylation, and 4 released inorganic phosphate with near-100% stoichiometry. Time-resolved infrared spectroscopy of the photolysis reaction, coupled with density functional theory calculations of vibrational frequencies, enabled us to infer a mechanism for the photolytic pathway, although there was some evidence for a second pathway also being operative. In contrast to the results for 4, photolysis of 5 appeared to release little or no monomethyl phosphate.


Subject(s)
Carboxylic Acids/chemistry , Phenylacetates/chemistry , Chromatography, Ion Exchange , Hot Temperature , Photochemistry
9.
J Neurosci Methods ; 180(1): 9-21, 2009 May 30.
Article in English | MEDLINE | ID: mdl-19427524

ABSTRACT

Rapid, localised photolytic release of neurotransmitters from caged precursors at synaptic regions in the extracellular space is greatly hampered at irradiation wavelengths in the near-UV, close to the wavelength of maximum absorption of the caged precursor, because of inner-filtering by strong absorption of light in the cage solution between the objective and cell. For this reason two-photon excitation is commonly used for photolysis, particularly at multiple points distributed over large fields; or, with near-UV, if combined with local perfusion of the cage. These methods each have problems: the small cross-sections of common cages with two-photon excitation require high cage concentrations and light intensities near the phototoxic limit, while local perfusion gives non-uniform cage concentrations over the field of view. Single-photon photolysis at 405 nm, although less efficient than at 330-350 nm, with present cages is more efficient than two-photon photolysis. The reduced light absorption in the bulk cage solution permits efficient wide-field uncaging at non-toxic intensities with uniform cage concentration. Full photolysis of MNI-glutamate with 100 micros pulses required intensities of 2 mW microm(-2) at the preparation, shown to be non-toxic with repeated exposures. Light scattering at 405 nm was estimated as 50% at 18 microm depth in 21-day rat cerebellum. Methods are described for: (1) varying the laser spot size; (2) photolysis calibration in the microscope with the caged fluorophore NPE-HPTS over the wavelength range 347-405 nm; and (3) determining the point-spread function of excitation. Furthermore, DM-Nitrophen photolysis at 405 nm was efficient for intracellular investigations of Ca2+-dependent processes.


Subject(s)
Electrophysiology/methods , Lasers/adverse effects , Light/adverse effects , Photochemistry/methods , Photolysis/radiation effects , Acetates/chemistry , Animals , Calcium Signaling/physiology , Calibration , Cerebellum/physiology , Ethylenediamines/chemistry , Glutamates/chemistry , Glutamates/radiation effects , Indoles/chemistry , Indoles/radiation effects , Organ Culture Techniques , Patch-Clamp Techniques/methods , Photic Stimulation/adverse effects , Photic Stimulation/methods , Rats , Rats, Sprague-Dawley , Synaptic Transmission/physiology
10.
J Neurosci Methods ; 181(2): 159-69, 2009 Jul 30.
Article in English | MEDLINE | ID: mdl-19422852

ABSTRACT

Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABA(A) receptors in neuronal compartments, the activation kinetics and pharmacology of GABA(A) receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABA(A) receptors with much lower affinity, reducing peak GABA-evoked responses with an IC(50) of approximately 0.5 mM. Most importantly, the kinetics of receptor activation, determined as 10-90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABA(A) receptors. With a laser spot of 1 microm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2 microm laterally and 7.5 microm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABA(A) receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.


Subject(s)
Cerebellum/physiology , Hippocampus/physiology , Indoles , Neurons/physiology , Organophosphonates , Photolysis , Receptors, GABA-A/metabolism , Synaptic Transmission , Animals , Cells, Cultured , Cerebellum/metabolism , Electrophysiology , Hippocampus/cytology , Hippocampus/metabolism , Indoles/radiation effects , Lasers , Neural Inhibition , Neurons/metabolism , Organophosphonates/radiation effects , Rats , Rats, Sprague-Dawley , Rats, Wistar , gamma-Aminobutyric Acid
11.
Biophys J ; 96(8): 3281-94, 2009 Apr 22.
Article in English | MEDLINE | ID: mdl-19383472

ABSTRACT

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P approximately NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12 degrees C of 10(5) M(-1) s(-1). This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P approximately NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa(2+) of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 +/- 0.6 microM and 4.7 +/- 1.5 microM at 12 and 20 degrees C, respectively. The rates of P approximately NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s(-1) at 12 and 20 degrees C, respectively. The time courses of isometric force and P approximately NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P(i) and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s(-1)) in the cross-bridge turnover during isometric contraction. At 12 degrees C, the A.M.ADP.P(i) and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20 degrees C, the force-bearing A.M.ADP.P(i) state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P approximately NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the P(i) transient during force recovery at 20 degrees C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.


Subject(s)
Adenosine Diphosphate/metabolism , Muscle Contraction/physiology , Muscle Fibers, Skeletal/metabolism , Animals , Calcium/metabolism , Computer Simulation , Coumarins , Female , Fluorescence , Kinetics , Linear Models , Muscle Strength , Mutation, Missense , Nucleoside-Diphosphate Kinase/genetics , Phosphorylation , Protein Isoforms/metabolism , Psoas Muscles/metabolism , Rabbits
12.
J Am Chem Soc ; 130(50): 17120-8, 2008 Dec 17.
Article in English | MEDLINE | ID: mdl-19053408

ABSTRACT

Replica exchange molecular dynamics (REMD) calculations were used to determine the conformation and dynamics of bifunctional rhodamine probes attached to pairs of cysteines in three model systems: (a) a polyalanine helix, (b) the isolated C helix (residues 53-66) of troponin C, and (c) the C helix of the N-terminal region (residues 1-90) of troponin C (sNTnC). In each case, and for both diastereoisomers of each probe-protein complex, the hydrophobic face of the probe is close to the protein surface, and its carboxylate group is highly solvated. The visible-range fluorescence dipole of the probe is approximately parallel to the line joining the two cysteine residues, as assumed in previous in situ fluorescence polarization studies. The independent rotational motion of the probe with respect to the protein on the nanosecond time scale is highly restricted, in agreement with data from fluorescence polarization and NMR relaxation studies. The detailed interaction of the probe with the protein surface depends on steric factors, electrostatic and hydrophobic interactions, hydrogen bonds, and hydration effects. The interaction is markedly different between diastereoisomers, and multiple preferred conformations exist for a single diasteroisomer. These results show that the combination of the hydrophobic xanthylium moiety of bifunctional rhodamine with the carboxylate substitution in its pendant phenyl ring causes the probe to be immobilized on the protein surface, while the two-site cysteine attachment defines the orientation of its fluorescence dipole. These features allow the orientation of protein components to be accurately determined in situ by polarized fluorescence measurements from bifunctional rhodamine probes.


Subject(s)
Molecular Conformation , Molecular Probes/chemistry , Rhodamines/chemistry , Troponin C/chemistry , Animals , Chickens , Computer Simulation , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Peptides/chemistry , Surface Properties , Time Factors
13.
Photochem Photobiol Sci ; 7(4): 423-32, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18385884

ABSTRACT

A new version of a benzophenone antenna-sensitised photolabile derivative of l-glutamate, which has a dicarboxylic acid substituent on the benzophenone to promote water solubility, has been synthesised. It does not show problems of precipitation in the presence of calcium ions that were encountered with related compounds in which one or two phosphate groups were present as water-solubilising substituents but retains the enhanced photolytic efficiency that results from the benzophenone antenna. Photolysis of the compound proceeds with stoichiometric release of l-glutamate and pharmacological evaluations have shown that the compound itself has no evidence of agonist or antagonist activity in its unphotolysed form.


Subject(s)
Calcium/chemistry , Glutamic Acid/chemistry , Indoles/chemistry , Neurosciences/methods , Nitro Compounds/chemistry , Animals , Cells, Cultured , Electrophysiology , Ions/chemistry , Molecular Structure , Patch-Clamp Techniques , Photochemistry , Rats , Rats, Sprague-Dawley , Sensitivity and Specificity , Solubility , Spectrophotometry, Infrared
14.
J Am Chem Soc ; 130(8): 2602-9, 2008 Feb 27.
Article in English | MEDLINE | ID: mdl-18251471

ABSTRACT

Fluorescence polarization measurements of bifunctional rhodamine (BR) probes provide a powerful approach to determine the in situ orientation of proteins within ordered complexes such as muscle fibers. For accurate interpretation of fluorescence measurements, it is important to understand the probe dynamics relative to the protein to which it is attached. We previously determined the structure of the N-domain of chicken skeletal troponin C, BR-labeled on the C helix, in complex with the switch region of troponin I, and demonstrated that the probe does not perturb the structure or dynamics of the protein. In this study, the motion of the fluorescence label relative to the protein has been characterized using NMR relaxation measurements of 13C-labeled methyl groups on the BR probe and 15N-labeled backbone amides of the protein. Probe dynamics were monitored using off-resonance 13C-R(1rho), 13C-R(1) and {1H}-13C NOE at magnetic field strengths of 500, 600, and 800 MHz. Relaxation data were interpreted in terms of the overall rotational correlation time of the protein and a two-time scale model for internal motion of the BR methyl groups, using a numerical optimization with Monte Carlo parameter error estimation. The analysis yields a 1.5 +/- 0.4 ps correlation time for rotation around the three-fold methyl symmetry axis, and a 0.8 +/- 0.4 ns rotational correlation time for reorientation of the 13C-14N bond with an associated S2s of 0.79 +/- 0.03. Order parameters of the backbone NH vectors in the helix to which the probe is attached average S2 approximately 0.85, implying that the amplitude of independent reorientation of the BR probe is small in magnitude, consistent with results from fluorescence polarization measurements in reconstituted muscle fibers.


Subject(s)
Fluorescent Dyes/chemistry , Magnetic Resonance Spectroscopy/methods , Rhodamines/chemistry , Troponin C/chemistry , Magnetic Resonance Spectroscopy/standards , Models, Molecular , Molecular Structure , Reference Standards
15.
Photochem Photobiol Sci ; 7(1): 84-97, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18167601

ABSTRACT

Photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds, studied here by time-resolved IR and UV spectroscopy, involves at least two pathways. In one, a conventional 2-nitrobenzyl type rearrangement takes place to release the photoprotected species via rapid decay of an aci-nitro intermediate. The alpha-carboxylate moiety of the CNB group is retained and the final by-product from this pathway is 2-nitrosophenylglyoxylate. Direct measurements of product formation confirmed that release via this pathway is faster for CNB-caged compounds than for related caged compounds without an alpha-carboxylate substituent and a rationale for the faster release rate is proposed. In a second pathway, photodecarboxylation of the starting material occurs: this pathway leads only to a slow, minor release of the photoprotected species. The extent to which the latter pathway contributes is affected by the nature of buffer salts in the irradiated solution. It was more prominent in an amine-based buffer (MOPS) than in phosphate buffer.


Subject(s)
Carboxylic Acids/chemistry , Nitrobenzenes/chemistry , Photolysis , Carbon Dioxide/chemistry , Decarboxylation , Hydrogen-Ion Concentration , Kinetics , Molecular Structure , Spectrophotometry
16.
J Biol Chem ; 283(2): 766-73, 2008 Jan 11.
Article in English | MEDLINE | ID: mdl-17965414

ABSTRACT

Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. (2006) Biochemistry 45, 13035-13045). The kinetics of ADP and deac-aminoADP dissociation from actomyosin V-HMM, following the power stroke, were determined using double-mixing stopped-flow fluorescence. These used either deac-aminoATP as the substrate with ADP or ATP chase or alternatively ATP as the substrate with either a deac-aminoADP or deac-aminoATP chase. Both sets of experiments show that the observed rate of ADP or deac-aminoADP dissociation from the trail head of actomyosin V-HMM is the same as from actomyosin V-S1. The dissociation of ADP from the lead head is decreased by up to 250-fold.


Subject(s)
Actomyosin/metabolism , Adenosine Diphosphate/metabolism , Myosin Type V/metabolism , Actins/isolation & purification , Actins/metabolism , Animals , Hydrolysis , Kinetics , Mice , Muscle, Skeletal/metabolism , Myosin Subfragments/metabolism , Rabbits , Spectrometry, Fluorescence
17.
Biophys J ; 93(3): 1008-20, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17483167

ABSTRACT

As part of a program to develop methods for determining protein structure in situ, sTnC was labeled with a bifunctional rhodamine (BR or BSR), cross-linking residues 56 and 63 of its C-helix. NMR spectroscopy of the N-terminal domain of BSR-labeled sTnC in complex with Ca(2+) and the troponin I switch peptide (residues 115-131) showed that BSR labeling does not significantly affect the secondary structure of the protein or its dynamics in solution. BR-labeling was previously shown to have no effect on the solution structure of this complex. Isometric force generation in isolated demembranated fibers from rabbit psoas muscle into which BR- or BSR-labeled sTnC had been exchanged showed reduced Ca(2+)-sensitivity, and this effect was larger with the BSR label. The orientation of rhodamine dipoles with respect to the fiber axis was determined by polarized fluorescence. The mean orientations of the BR and BSR dipoles were almost identical in relaxed muscle, suggesting that both probes accurately report the orientation of the C-helix to which they are attached. The BSR dipole had smaller orientational dispersion, consistent with less flexible linkers between the rhodamine dipole and cysteine-reactive groups.


Subject(s)
Rhodamines/chemistry , Troponin C/chemistry , Animals , Carbon Isotopes , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Muscle Fibers, Skeletal/chemistry , Muscle Fibers, Skeletal/ultrastructure , Muscle, Skeletal/chemistry , Muscle, Skeletal/ultrastructure , Protein Binding , Protein Conformation , Solvents
18.
Biochemistry ; 46(12): 3872-80, 2007 Mar 27.
Article in English | MEDLINE | ID: mdl-17311416

ABSTRACT

Na+-Dependent transmembrane transport of small neutral amino acids, such as glutamine and alanine, is mediated, among others, by the neutral amino acid transporters of the solute carrier 1 [SLC1, alanine serine cysteine transporter 1 (ASCT1), and ASCT2] and SLC38 families [sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4]. Many mechanistic aspects of amino acid transport by these systems are not well-understood. Here, we describe a new photolabile alanine derivative based on protection of alanine with the 4-methoxy-7-nitroindolinyl (MNI) caging group, which we use for pre-steady-state kinetic analysis of alanine transport by ASCT2, SNAT1, and SNAT2. MNI-alanine has favorable photochemical properties and is stable in aqueous solution. It is also inert with respect to the transport systems studied. Photolytic release of free alanine results in the generation of significant transient current components in HEK293 cells expressing the ASCT2, SNAT1, and SNAT2 proteins. In ASCT2, these currents show biphasic decay with time constants, tau, in the 1-30 ms time range. They are fully inhibited in the absence of extracellular Na+, demonstrating that Na+ binding to the transporter is necessary for induction of the alanine-mediated current. For SNAT1, these transient currents differ in their time course (tau = 1.6 ms) from previously described pre-steady-state currents generated by applying steps in the membrane potential (tau approximately 4-5 ms), indicating that they are associated with a fast, previously undetected, electrogenic partial reaction in the SNAT1 transport cycle. The implications of these results for the mechanisms of transmembrane transport of alanine are discussed. The new caged alanine derivative will provide a useful tool for future, more detailed studies of neutral amino acid transport.


Subject(s)
Alanine/metabolism , Alanine/pharmacology , Amino Acid Transport Systems, Neutral/metabolism , Sodium/metabolism , Alanine/analogs & derivatives , Cell Line , Humans , Ion Transport/drug effects , Ion Transport/physiology , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Photolysis
19.
Bioconjug Chem ; 18(1): 231-7, 2007.
Article in English | MEDLINE | ID: mdl-17226977

ABSTRACT

Two fluorescent conjugates of sialic acid have been prepared, with a convenient synthetic route that involves preparation of an unsaturated benzyl ester by cross-metathesis, followed by combined hydrogenation/ hydrogenolysis to provide a sialoside bearing a delta-carboxybutyl group, suitable for coupling with the chosen fluorophores. The fluorescent conjugates bound to bromelain-cleaved hemagglutinin (BHA) with affinities in the low microM range. Binding was accompanied by approximately 4.5-fold fluorescence enhancement for the dansyl conjugate 1 and approximately 3-fold fluorescence quenching for the pyrene conjugate 3.


Subject(s)
Fluorescent Dyes/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/analysis , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Influenza A virus/chemistry , Ligands , Molecular Structure
20.
Biochemistry ; 45(49): 14764-71, 2006 Dec 12.
Article in English | MEDLINE | ID: mdl-17144669

ABSTRACT

A novel biosensor for inorganic phosphate (Pi) has been developed based on the phosphate binding protein of Escherichia coli. Two cysteine mutations were introduced and labeled with 6-iodoacetamidotetramethylrhodamine. When physically close to each other and correctly oriented, two rhodamine dyes interact to form a noncovalent dimer. In this state, they have little or no fluorescence, unlike the high fluorescence intensity of monomeric rhodamine. The labeling sites were so placed that the distance and orientation between the rhodamines change as a consequence of the conformational change associated with Pi binding. This movement alters the extent of interaction between the dyes. The best mutant of those tested (A17C, A197C) gives rise on average to approximately 18-fold increase in fluorescence intensity as Pi binds. The kinetics of interaction with Pi were measured at 10 degrees C. Under these conditions, the fluorescence increase associated with Pi binding has a maximum rate of 267 s-1. The Pi dissociation rate is 6.6 s-1, and the dissociation constant is 70 nM. An application of the sensor is demonstrated for measuring ATP hydrolysis in real time as a helicase moves along DNA. Advantages of the new sensor are discussed, both in terms of the use of a rhodamine fluorophore and the potential of this double labeling strategy.


Subject(s)
Phosphate-Binding Proteins/metabolism , Phosphates/analysis , Rhodamines , Biosensing Techniques , Cysteine , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Kinetics , Mutagenesis, Site-Directed , Phosphate-Binding Proteins/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Spectrophotometry
SELECTION OF CITATIONS
SEARCH DETAIL
...