Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.
AuthorsBorejdo J, Ushakov DS, Akopova I
JournalBiophys J
PubMed ID12023239
'Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the ... More
E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics.
'Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca(2+)-binding site. We have studied transgenic (Tg) mouse ... More
Orientational changes of crossbridges during single turnover of ATP.
AuthorsBorejdo J, Akopova I
JournalBiophys J
PubMed ID12668452
'Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the alpha-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added ... More
Flash photolysis studies of excitation-contraction coupling, regulation, and contraction in smooth muscle.
AuthorsSomlyo AP, Somlyo AV
JournalAnnu Rev Physiol
PubMed ID2184779
'1. Flash photolysis of caged compounds of phenylephrine, inositol 1, 4, 5 trisphosphate (InsP3), GTP gamma S, ATP, and CTP has been successfully used to study excitation-contraction coupling, contractile regulation, and contraction in smooth muscle. Major processes explored with this method were (a) the delay between agonist-receptor interaction and contraction ... More
Inactivation of P2X2 purinoceptors by divalent cations.
AuthorsDing S, Sachs F
JournalJ Physiol
PubMed ID10639098
'1. P2X2 channels are activated by extracellular ATP. Despite being commonly described as non-desensitizing, P2X2 receptors do desensitize or inactivate. In the unspliced, 472 amino acid isoform of the P2X2 receptor, inactivation required membrane disruption and the presence of extracellular Ca2+. 2. The ability to inactivate whole-cell currents developed slowly ... More
Properties and uses of photoreactive caged compounds.
AuthorsMcCray JA, Trentham DR
JournalAnnu Rev Biophys Biophys Chem
PubMed ID2660825
Fluorescence imaging of Na+ influx via P2X receptors in cochlear hair cells.
AuthorsHousley GD, Raybould NP, Thorne PR
JournalHear Res
PubMed ID9641314
'The adenosine 5''-triphosphate (ATP)-activated membrane conductance, mediated by P2X receptors, was examined in isolated guinea-pig cochlear inner and outer hair cells. Photo-activated release of caged-ATP elicted a 30-ms latency inwardly rectifying non-selective cation conductance, blocked by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS; 10-100 microM), consistent with the direct activation ... More
Correlation between mechanical and enzymatic events in contracting skeletal muscle fiber.
AuthorsShepard A, Borejdo J
JournalBiochemistry
PubMed ID15005615
'The conventional hypothesis of muscle contraction postulates that the interaction between actin and myosin involves tight coupling between the power stroke and hydrolysis of ATP. However, some in vitro experiments suggested that hydrolysis of a single molecule of ATP caused multiple mechanical cycles. To test whether the tight coupling is ... More
Simultaneous measurement of rotations of myosin, actin and ADP in a contracting skeletal muscle fiber.
AuthorsShepard AA, Dumka D, Akopova I, Talent J, Borejdo J
JournalJ Muscle Res Cell Motil
PubMed ID15711885
'The rotation of myosin heads and actin were measured simultaneously with an indicator of the enzymatic activity of myosin. To minimize complications due to averaging of signals from many molecules, the signal was measured in a small population residing in a femtoliter volume of a muscle fiber. The onset of ... More
Photolysis of caged phosphatidic acid induces flagellar excision in Chlamydomonas.
AuthorsGoedhart J, Gadella TW
JournalBiochemistry
PubMed ID15065870
'Phosphatidic (PtdOH) acid formation is recognized as an important step in numerous signaling pathways in both plants and mammals. To study the role of this lipid in signaling pathways, it is of major interest to be able to increase the amount of this lipid directly. Therefore, "caged" PtdOH was synthesized, ... More
Caged compounds: photorelease technology for control of cellular chemistry and physiology.
AuthorsEllis-Davies GC
JournalNat Methods
PubMed ID17664946
Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies ... More
Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other ... More
Illuminating the chemistry of life: design, synthesis, and applications of
AuthorsLee HM, Larson DR, Lawrence DS,
JournalACS Chem Biol
PubMed ID19298086
Biological systems are characterized by a level of spatial and temporal organization that often lies beyond the grasp of present day methods. Light-modulated bioreagents, including analogs of low molecular weight compounds, peptides, proteins, and nucleic acids, represent a compelling strategy to probe, perturb, or sample biological phenomena with the requisite ... More
Inhibition of ATP-regulated K(+)-channels by a photoactivatable ATP-analogue in mouse pancreatic beta-cells.
AuthorsAmmälä C, Bokvist K, Galt S, Rorsman P
JournalBiochim Biophys Acta
PubMed ID2049403
The effects of a photoactivable (DMNPE-caged) ATP-analogue on ATP-regulated K(+)-channels (KATP-channel) in mouse pancreatic beta-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a Ki of 17 microM; similar to the 11 microM obtained for standard ... More
Kinetics and mechanism of DNA repair. Evaluation of caged compounds for use in studies of u.v.-induced DNA repair.
AuthorsMeldrum RA, Shall S, Wharton CW
JournalBiochem J
PubMed ID2327973
Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the gamma-phosphate are placed in situ by permeabilizing cells ... More
Modulation of ATP-sensitive potassium channel activity by flash-photolysis of 'caged-ATP' in rat heart cells.
AuthorsNichols CG, Niggli E, Lederer WJ
JournalPflugers Arch
PubMed ID2315010
We have used 'caged-ATP' to investigate the kinetic behavior of KATP channels in ventricular cells from rat heart. In whole cells, loaded with 'caged-ATP', an increase of intracellular [ATP] following a UV light flash produced a decrease of KATP channel current that was too slow (tau approximately 300 ms) to ... More
Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution.
AuthorsBernardinelli Y, Haeberli C, Chatton JY
JournalCell Calcium
PubMed ID15862347
Flash photolysis has become an essential technique for dynamic investigations of living cells and tissues. This approach offers several advantages for instantly changing the concentration of bioactive compounds outside and inside living cells with high spatial resolution. Light sources for photolysis need to deliver pulses of high intensity light in ... More
Structural features of nonpeptide prenyl pyrophosphates that determine their antigenicity for human gamma delta T cells.
AuthorsMorita CT, Lee HK, Wang H, Li H, Mariuzza RA, Tanaka Y
JournalJ Immunol
PubMed ID11418629
Human Vgamma2Vdelta2(+) T cells proliferate in vivo during many microbial infections. We have found that Vgamma2Vdelta2(+) T cells recognize nonpeptide prenyl pyrophosphates and alkylamines. We now have defined structural features that determine the antigenicity of prenyl pyrophosphates by testing synthetic analogs for bioactivity. We find that the carbon chain closest ... More
Targeting expression with light using caged DNA.
AuthorsMonroe WT, McQuain MM, Chang MS, Alexander JS, Haselton FR
JournalJ Biol Chem
PubMed ID10409633
In this report, we describe the inactivation and site-specific light induction of plasmid expression using a photosensitive caging compound. Plasmids coding for luciferase were caged with 1-(4, 5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) and transfected into approximately 1-cm diameter sites of the skin of rats with particle bombardment. Skin sites transfected with caged plasmids ... More
Control of DNA hybridization with photocleavable adducts.
AuthorsGhosn B, Haselton FR, Gee KR, Monroe WT
JournalPhotochem Photobiol
PubMed ID15869326
Previous reports have shown that 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE-caged oligonucleotides have changed spectrophotometric and electrophoretic properties that can ... More
Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces.
AuthorsIshijima A, Kojima H, Higuchi H, Harada Y, Funatsu T, Yanagida T
JournalBiophys J
PubMed ID8770215
We have developed a new technique for measurements of piconewton forces and nanometer displacements in the millisecond time range caused by actin-myosin interaction in vitro by manipulating single actin filaments with a glass microneedle. Here, we describe in full the details of this method. Using this method, the elementary events ... More
Photochemical gating of heterologous ion channels: remote control over genetically designated populations of neurons.
AuthorsZemelman BV, Nesnas N, Lee GA, Miesenbock G
JournalProc Natl Acad Sci U S A
PubMed ID12540832
Heterologous proteins capable of transducing physical or chemical stimuli into electrical signals can be used to control the function of excitable cells in intact tissues or organisms. Restricted genetically to circumscribed populations of cellular targets, these selectively addressable sources of depolarizing current can supply distributed inputs to neural circuits, stimulate ... More