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G Marriott

Publications and source records attributed to G Marriott.

33 records · Page 2Linked to original sources
PubMed1996

Light-directed generation of the actin-activated ATPase activity of caged heavy meromyosin.

An understanding of the molecular mechanism of muscle contraction will require a complete description of the kinetics of the myosin motor in vitro and in vivo. To this end chemical relaxation studies employing light-directed generation of ATP from caged ATP have provided detailed kinetic information in muscle fibers. A more direct approach would be to trigger the actin-activated ATPase activity from a caged myosin, i.e., myosin whose activity is blocked upon derivatization with a photolabile protection group. Herein we report that a new type of caged reagent can be used to prepare a caged heavy meromyosin by modification of critical thiol groups, i.e., a chemically modified motor without activity that can be reactivated at will using a pulse of near-ultraviolet light. Heavy meromyosin modified at Cys-707 with the thiol reactive reagent 1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene does not exhibit an actin-activated ATPase activity and may be viewed as a caged protein. Absorption spectroscopy showed that the thioether bond linking the cage group to Cys-707 is cleaved following irradiation (340-400 nm) via a transient aci-nitro intermediate which has an absorption maximum at 440 nm and decays with a rate constant of 45.6 s(-1). The in vitro motility assay showed that caged heavy meromyosin cannot generate the force necessary to move actin filaments although following irradiation of the image field with a 30 ms pulse of 340-400 nm light the caged group was removed with the concomitant movement of most filaments at a velocity of 0.5-2 micron/s compared to 3-4 micron/s for unmodified HMM. The specificity and simplicity of labeling myosin with the caged reagent should prove useful in studies of muscle contraction in vivo.

Actins↗
PubMed1995

Proximity relationships and structural dynamics of the phalloidin binding site of actin filaments in solution and on single actin filaments on heavy meromyosin.

Distance relationships between phalloidin binding sites on F-actin have been investigated using fluorescence resonance energy transfer (FRET) techniques in solution and on single F-actin filaments bound to heavy meromyosin (HMM). Filaments saturated with an equimolar concentration of fluoresceinisothiocyanatophalloidin (FITC-ph) as the donor and tetramethylrhodamineisothiocyanatophalloidin (TRITC-ph) as the acceptor and control filaments saturated with either FITC-ph or TRITC-ph were characterized by absorption and fluorescence spectroscopy and the in vitro motility assay. Fluorescence excitation polarization spectroscopy showed hetero-FRET occurred within colabeled filaments, whereas homo-FRET was observed in control filaments. The distance measured between adjacent phalloidin binding sites using randomly labeled FITC-ph and TRITC-ph was 37.2 A using steady-state spectral analysis and 36.9 A using time-resolved spectroscopy with a radial coordinate of 14.5 A. Measurements of the distance between fluorescent phalloidin groups using the atomic model of F-actin [Lorenz, M., Popp, D., & Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836] suggest transfer occurs between adjacent phalloidin molecules on opposite actin helices (39 A), rather than between adjacent phalloidins along the same actin helix (55.4 A). A quantitative fluorescence microscope technique was described that measures the proximity of adjacent FITC-ph and TRITC-ph on single filaments immobilized on HMM. Here a distance of 36.2 A was calculated which was unchanged during ATP-dependent sliding of F-actin on HMM. Spatially resolved FRET measurements are being used to observe the effects of actin binding proteins on the structural properties along the length of single actin filaments.

Actins↗
PubMed1994

Caged protein conjugates and light-directed generation of protein activity: preparation, photoactivation, and spectroscopic characterization of caged G-actin conjugates.

A simple method is described to prepare caged (inactive) protein complexes using the amino group-directed photo-deprotection group [(nitroveratryl)oxy]chlorocarbamate (NVOC-Cl). In this study, I show how the polymerization activity of G-actin in physiological salt solution is lost upon conjugation of essential lysine residues of G-actin with NVOC-Cl. Reaction conditions were optimized to prepare caged G-actin in high yield, and the conjugate was characterized by biochemical and absorption spectroscopic methods. Upon excitation of caged G-actin in physiological salt solutions with near-ultraviolet light, an efficient photo-deprotection reaction occurs via photoisomerization of the (nitrophenyl)ethyl group of NVOC, which results in cleavage of the carbamate linkage between the protection reagent and G-actin. A standard irradiation condition was then defined which leads to photoactivation of F-actin from caged G-actin with a yield of more than 90%. Photoactivated F-actin was characterized according to its sedimentation behavior, electron microscopic analysis, and sliding velocity on heavy meromyosin determined with the in vitro motility assay. The results of these assays were similar to those obtained from unmodified F-actin. I also report the preparation of caged G-actin conjugated at cysteine 374 with tetramethylrhodamine iodoacetamide and caged fluorescein maleimide. These caged G-actin conjugates can be used to generate fluorescent, polymerization competent G-actin following near-ultraviolet irradiation. Given the widespread applications of caged substrates and ligands in cell biology, the simple method described herein to prepare and photoactivate caged protein conjugates is expected to advance investigations on the regulation of protein activity in living cells.

Actins↗
PubMed1994

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found. Through the coupling of SBMC to streptavidin,a plethora of biotin-based tracer molecules are available for immunocytochemical studies.

Animals↗
PubMed1993

Orientation of actin monomers in moving actin filaments.

We have visualized, under an optical microscope, the orientations of actin monomers in individual actin filaments undergoing Brownian motion in solution, actively sliding past myosin molecules, or immobile on a surface. For the visualization, two strategies have been adopted. One is to exploit the fluorescence polarization of a fluorescent probe firmly attached to actin. Using the probe phalloidin-tetramethylrhodamine, the fluorescence was clearly polarized along the filament axis, showing alignment of the probe molecules along the filament axis. Within our temporal resolution of 33 ms and spatial resolution of better than 1 micron (average over approximately 10(2) actin monomers), the orientation of the probe (hence of actin monomers) did not change upon interaction of the filament with heavy meromyosin; myosin-induced reorientation was estimated to be a few degrees at most. This first method, while highly sensitive to small reorientations of monomers off or toward the filament axis, does not report on reorientations around the axis. To detect rotation around the filament axis, we adopted the second strategy in which we attached small plastic beads to the actin filaments. Axial turns would be immediately apparent from the movement of the beads. Preliminary observations indicate that actin filaments can slide over a heavy meromyosin-coated surface without axial rotations. Since rotations have been implicated in different experiments, we are currently investigating the source of the apparent discrepancy. The attached bead also serves as a handle through which we can apply force, via optical tweezers, on the filament. By letting the sliding actin filament pull the bead against the optical force, we were able to estimate the sliding force and its fluctuation.

Actin Cytoskeleton↗
PubMed1992

Photomodulation of the nucleating activity of a photocleavable crosslinked actin dimer.

The ability to generate substrate concentration jumps through photo-deprotection of amine, carboxyl and phosphate groups has been an important development for investigations of protein activity in complex systems. To broaden the versatility and applications of photo-deprotection techniques for the photomodulation of protein activity we describe the synthesis and characterisation of a reagent for generating free thiol from thioether groups and a related photocleavable, heterobifunctional crosslinking reagent. Chemical and spectroscopic studies of a model thiol protected derivative were used to show some features of thiol group photodeprotection. To demonstrate how the photocleavable crosslinking reagent may be used to modulate the activity of proteins we investigated the effect of light on the nucleating activity of crosslinked actin dimer; thus following near-ultraviolet irradiation of the actin dimer the crosslink was cleaved, presumeably at the thioether bond, resulting in the concomitant dissociation of dimer, loss of nucleating activity and creation of a concentration jump of polymerisable G-actin monomer. On the basis of this initial study we discuss applications and limitations of these reagents for the photomodulation of protein activity in vitro and in vivo.

Actins↗
PubMed1991

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.

3T3 Cells↗
PubMed1990

Absorption and fluorescence spectroscopic studies of the Ca2(+)-dependent lipid binding protein p36: the annexin repeat as the Ca2+ binding site.

The existence of a single tryptophan residue in the protein p36, a member of a recently characterized family of Ca2+ binding proteins called annexins, is exploited to provide unique spectroscopic information on the annexin repeat motif and its role in Ca2+ binding. The differences in ultraviolet absorption and fluorescence excitation upon Ca2+ binding are interpreted solely in terms of this tryptophan, which, in view of the pronounced blue-shifts and the presence of vibronic structure, seems to reside in a highly nonpolar environment. The fluorescence emission from the protein is correspondingly blue-shifted, and it is found to transfer energy in resonance with Tb3+ absorption lines in the near-ultraviolet. This effect allows us to locate the Tb3+ and, by implication, the Ca2+ binding site to within ca. 8 A of the tryptophan residue.

Animals↗
PubMed1988

Going back to basics.

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Centralized Hospital Services↗
PubMed1988

Spectroscopic and functional characterization of an environmentally sensitive fluorescent actin conjugate.

Rabbit skeletal muscle F-actin has been selectively labeled at a cysteine residue with the environmentally sensitive fluorophore 6-acryloyl-2-(dimethylamino)naphthalene. The fluorescent actin conjugate behaves similarly to native actin with respect to the polymerization kinetics, critical monomer concentration, and ability to form F-actin paracrystals. Upon polymerization to F-actin, the absorption of the actin conjugate is red-shifted, whereas the fluorescence emission is blue-shifted 740 wavenumbers and is accompanied by a decrease in the fluorescence bandwidth of 470 wavenumbers. These large shifts in the spectral properties of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) in actin provide a simple method for obtaining a spectral discrimination between the G- and F-actin populations during the polymerization reaction. Steady-state fluorescence techniques were used to study the environment of the fluorophore in the monomeric and polymeric forms of actin. Fluorescence emission spectral analysis and quenching and polarization studies of G-actin-Prodan indicated that the fluorophore lies immobile on the protein surface but with one of its faces in full contact with the solvent. In F-actin, the fluorophore has a limited exposure to the solvent and is located in a dielectric environment similar to those seen for Prodan in polar, aprotic solvents or buried within a protein matrix [Macgregor, R. B., Jr., & Weber, G. (1986) Nature (London) 318, 70-73]. Additionally, our results demonstrate that the Prodan molecule conjugated to F-actin is completely immobile during its fluorescence lifetime, exhibits an increase in the resonance energy transfer (RET) from tryptophan residues compared to that observed in G-actin, and shows evidence of homologous RET within the polymer.

2-Naphthylamine↗
PubMed1988

p36, the major cytoplasmic substrate of src tyrosine protein kinase, binds to its p11 regulatory subunit via a short amino-terminal amphiphatic helix.

Protein I is a hetero-tetramer which contains two copies each of p11 and p36. p36 (calpactin I, lipocortin II) is a major substrate of retrovirally encoded tyrosine protein kinases, while p11 modulates several Ca2+-induced properties also displayed by p36 alone. Here we have characterized the p11 binding site on p36 by fluorescence spectroscopy using porcine p36 labelled at cysteine 8 with the fluorophore Prodan (6-proprionyl-2-dimethylamino-naphthalene). We have used peptides of differing length from the amino-terminal domain of p36 to restrict the major binding site to the first 12 residues. Noticeable binding is still observed with a peptide containing only the first nine residues. Interestingly the N-terminal acetyl group of p36 forms a functional part of the p11 binding site. CD studies indicate that the binding region can form an alpha-helix, which seems to have amphiphatic properties when projected on a helical wheel. This structural element is also known for a calmodulin binding protein. Thus the question is raised whether other p11/calmodulin-related proteins interact with their target proteins via a similar mechanism. We also discuss how p11 could modulate p36 associated properties.

Amino Acid Sequence↗
PubMed1977

Postoperative analgesia with ketamine and pethidine.

A double-blind trial compared ketamine with pethidine, pethidine alone at two dose levels and a placebo, in patients with postoperative pain. By assessment of pain intensity, observed relief and side-effects, the active drugs were clearly distinguishable from the placebo. With the doses used, however, the combination of ketamine with pethidine showed no advantage over pethidine alone.

Adult↗
PubMedDate not supplied

Preparation and photoactivation of caged fluorophores and caged proteins using a new class of heterobifunctional, photocleavable cross-linking reagents.

The design, synthesis, and spectroscopic and chemical properties of four members of a new class of heterobifunctional photocleavable (caged) cross-linking reagents were described. One of the two reactive groups of the cross-linker reacted with amino groups to form the corresponding photolabile carbamates. Amino group containing compounds or proteins caged with these reagents can be coupled through the thiol reactive oxirane group of the cross-linker to a different biomolecule or to a thiol-derivatized surface. The 3,4-dimethoxy-6-nitrophenyl photoisomerization group of the reagent was physically and chemically isolated from the cross-linking functionality, and the high extinction coefficient and red-shifted action spectrum of this chromophore make it suitable for photoactivation applications of caged compounds on surfaces or in living cells. The bifunctional, photocleavable cross-linking reagents were used to prepare a thiol reactive caged rhodamine 110. The new reagents and conjugation procedures described may be used as part of a general procedure to cage the activity of proteins by physically masking binding sites.

Actins↗