David R. Trentham

Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay

The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3‐l‐(2‐nitrophenyl)ethyl ester of ATP (NPE‐caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pisensitive probe, MDCC‐PBP, a coumarin‐labelled A197C mutant of the phosphate‐binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH‐linked coupled enzyme assay. On photolysis of NPE‐caged ATP in the presence of Ca2+ at 20°C, the fluorescence increase of MDCC‐PBP was non‐linear with time. ATPase activity was 41 s−1 in the first turnover based on a myosin subfragment 1 concentration of 150 μm. This was calculated from a linear regression of the fluorescence signal reporting 20‐150 μm of Pi release. Tension was at 67 % of its isometric level by the time 150 μm Pi was released. ATPase activities were 36 and 31 s−1for Pi released in the ranges of 150‐300μM and 300‐450 μm, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20°C. An NADH‐linked assay showed the ATPase activity had a lower limit of 12.7 s−1 at 20°C. The response to photoly tic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s−1. On photolysis of NPE‐caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s−1 at 20°C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl‐EGTA. At 5°C the ATPase rate was 5.8 and 4.0 s−1 in the first and second turnovers, respectively. These rates are comparable to those when NPE‐caged ATP was used. The influence of ADP and Pi on the ATPase activities was measured using the MDCC‐PBP and NADH‐linked assays, respectively. ADP (0.5 him) decreased the initial ATPase rate by 23%. Pi (10 him) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. The MDCC‐PBP assay and NPE‐caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5°C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s−1 during the first turnover (based on 180 μm myosin subfragment 1). Tension was 74% of its isometric level by the time 180 μm Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.

Orientation changes of the myosin light chain domain during filament sliding in active and rigor muscle

Structural changes in myosin power many types of cell motility including muscle contraction. Tilting of the myosin light chain domain (LCD) seems to be the final step in transducing the energy of ATP hydrolysis, amplifying small structural changes near the ATP binding site into nanometer-scale motions of the filaments. Here we used polarized fluorescence measurements from bifunctional rhodamine probes attached at known orientations in the LCD to describe the distribution of orientations of the LCD in active contraction and rigor. We applied rapid length steps to perturb the orientations of the population of myosin heads that are attached to actin, and thereby characterized the motions of these force-bearing myosin heads. During active contraction, this population is a small fraction of the total. When the filaments slide in the shortening direction in active contraction, the long axis of LCD tilts towards its nucleotide-free orientation with no significant twisting around this axis. In contrast, filament sliding in rigor produces coordinated tilting and twisting motions.

Kinetics of the conductance evoked by noradrenaline, inositol trisphosphate or Ca2+ in guinea-pig isolated hepatocytes.

1. Guinea‐pig hepatocytes respond to noradrenaline (NA, 5‐10 microM) with a large membrane conductance increase to K+ and Cl‐. The response has a long initial delay (range 2‐30 s). Following the delay, the K+ conductance (studied in Cl(‐)‐free solutions) rises quickly to a peak in 1‐2 s and is maintained in the continued presence of NA, though often with superimposed oscillations of conductance. The roles of intracellular Ca2+ and D‐myo‐inositol 1,4,5‐trisphosphate (InsP3) in this complex response have been investigated by rapid photolytic release of intracellular Ca2+ (from Nitr5‐Ca2+ buffers) or InsP3 from ‘caged’ InsP3. 2. A rapid increase of intracellular [Ca2+] produced an immediate membrane conductance increase which rose approximately exponentially to a new steady level, consistent with a direct activation of Ca2(+)‐dependent ion channels. 3. Following a pulse of InsP3, conductance rose after a brief delay (range 70‐1500 ms) which was shortest at high [InsP3] or if the initial cytosolic [Ca2+] had been raised above normal levels. The maximum conductance produced by InsP3 was similar in each cell to the peak recorded with NA and could be evoked by InsP3 concentrations of 0.5‐1 microM. 4. The rates of rise of conductance increased with InsP3 concentration in the range 0.25‐12.5 microM (range 10‐90%, rise times 90‐1000 ms), indicating that InsP3‐evoked Ca2(+)‐efflux from stores increases with InsP3 concentration in this range. 5. Photochemically released InsP3 and Ca2+ activate at physiological concentrations the same membrane conductances as NA. The results indicate that the long initial delay in NA action occurs prior to or during generation of InsP3. The mechanism of the delay and the subsequent apparently all‐or‐none conductance increase during NA action are discussed in terms of the high co‐operativity in InsP3 and Ca2+ actions and an additional positive feedback step. 6. Evidence was found of a negative interaction between [Ca2+] and InsP3‐evoked Ca2+ release. The time course of the recovery of InsP3‐evoked Ca2+ release following a rise of cytosolic [Ca2+] suggests that this interaction may be important in regulating oscillatory responses of [Ca2+] during hormonal stimulation of guinea‐pig hepatocytes.

[16] Synthesis and properties of caged nucleotides

Publisher Summary Photosensitive precursors have been termed “caged compounds.” This chapter focuses on caged compounds that yield nucleotides or nucleotide analogs on photolysis. In this approach, a biological preparation is equilibrated with a caged nucleotide, and it is then illuminated using a near-UV light pulse to liberate the nucleotide that initiates a biological response. Such a strategy has been used to make time-resolved measurements of the actions of ATP and ATPγS on muscle fibers, the Na+/K+ ion pump ATPase, and sarcoplasmic reticulum vesicles and of GTP analogs on sensory neurons. These nucleotides are common in that they contain weakly acidic phosphate groups and the photolabile precursors used are 1-(2-nitrophenyl)ethyl phosphate esters. The esters have advantageous photochemical properties. This chapter describes the synthesis and characterization of 1-(2-nitrophenyl)ethyl phosphate esters of nucleotides using as representative examples caged ATP, ATPβ, γNH, and GTPγS.

Postsynaptic activation at the squid giant synapse by photolytic release of L‐glutamate from a ‘caged’ L‐glutamate.

1. Pharmacological evidence suggests L‐glutamate is a strong candidate as a transmitter at the giant synapse of the squid. Postsynaptic activation at the giant synapse cannot be effected by conventional application of putative neurotransmitters by iontophoresis or perfusion, apparently because the complex structure of the synapse prevents a sufficiently rapid change in concentration at the postsynaptic membrane. Flash photolytic release of L‐glutamate from a pharmacologically inert ‘caged’ L‐glutamate pre‐equilibrated in the stellate ganglion of Alloteuthis or Loligo was used to determine whether L‐glutamate can produce postsynaptic activation when released rapidly in the synaptic clefts. 2. The preparation, reaction mechanism and properties of the caged L‐glutamate, N‐1‐(2‐nitrophenyl)ethoxycarbonyl‐L‐glutamate, are described. The product quantum yield on photolysis was 0.65 (+/‐ 0.05). On flash photolysis glutamate release followed a single exponential time‐course in the pH range 5.5‐7.8. The rate constant was proportional to [H+] and was 93 s‐1 at pH 5.5 and 16 degrees C in artificial sea water (ionic strength, I = 0.68 M). 3. At pH 7.8 flash photolysis of caged glutamate pre‐equilibrated in the synapse caused only a slow depolarization. A second photolytic release of L‐glutamate or transsynaptic activation produced no further depolarization, suggesting desensitization and inactivation of postsynaptic mechanisms by the initial pulse of L‐glutamate. 4. Synaptic transmission in the giant synapse was normal at pH 5.5. Flash photolysis at pH 5.5 caused rapid production of L‐glutamate within the synaptic cleft and a fast postsynaptic depolarization which generated postsynaptic action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ orientations of protein domains: troponin C in skeletal muscle fibers.

A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca(2+)-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Ca(2+)-free domain, and its D helix is approximately perpendicular to the actin filament. In contrast to crystal structures of isolated TnC, the D and E helices are not collinear. On muscle activation, the N lobe orientation becomes more disordered and the average angle between the C helix and the filament changes by 32 degrees +/- 5 degrees. These results illustrate the potential of in situ measurements of helix and domain orientations for elucidating structure-function relations in native macromolecular complexes.

Activation of NMDA receptors is necessary for the induction of associative long‐term potentiation in area CA1 of the rat hippocampal slice

1 It is commonly assumed that the role of the strongly activated heterosynaptic input during the induction of associative long‐term potentiation (LTP) is to relieve the magnesium blockade of NMDA receptors located at the weakly stimulated synapses and thereby allow the weak input to undergo potentiation. We tested this assumption by using a caged form of the NMDA receptor antagonist, d‐(–)‐2‐amino‐5‐phosphonopentanoic acid (d‐AP5) to block the activation of NMDA receptors at the weak input in a conditioning protocol for the induction of associative LTP in area CA1 of the rat hippocampal slice. 2 The effect of releasing d‐AP5 by flash photolysis of 100 μm caged d‐AP5 (N‐[l‐(2‐nitro‐phenyl)ethoxycarbonyl]‐d‐AP5) on pharmacologically isolated NMDA receptor‐mediated field EPSPs was examined in area CA1. The slope of the EPSP was reduced by 71% within 50 ms of the initiation of the photolytic reaction when the concentration of released d‐AP5 had reached 2.0‐2.5 μm and was reduced by 95% within 1 min (10 μmd‐AP5 released). 3 Associative LTP was induced by pairing a strong tetanus to one input with a weak tetanus (subthreshold for homosynaptic LTP) to a second input. The strong tetanus preceded the weak by 50 ms. Rapid application of d‐AP5, by flash photolysis of caged d‐AP5, coincident with the last shock of the strong tetanus, resulted in the blockade of NMDA receptor activation during the period of the weak tetanus. Associative LTP was blocked by photolysis of caged d‐AP5 but was normally expressed in experiments using caged l‐AP5. 4 We conclude that activation of NMDA receptors at the weakly activated input is an essential requirement for synaptically induced associative LTP.

Steady-state fluorescence polarization studies of the orientation of myosin regulatory light chains in single skeletal muscle fibers using pure isomers of iodoacetamidotetramethylrhodamine.

The regulatory light chain (RLC) from chicken gizzard myosin was covalently modified on cysteine 108 with either the 5- or 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLCs were purified by fast protein liquid chromatography and characterized by reverse-phase high-performance liquid chromatography (HPLC), tryptic digestion, and electrospray mass spectrometry. Labeled RLCs were exchanged into the native myosin heads of single skinned fibers from rabbit psoas muscle, and the ATR dipole orientations were determined by fluorescence polarization. The 5- and 6-ATR dipoles had distinct orientations, and model orientational distributions suggest that they are more than 20 degrees apart in rigor. In the rigor-to-relaxed transition (sarcomere length 2.4 microm, 10 degrees C), the 5-ATR dipole became more perpendicular to the fiber axis, but the 6-ATR dipole became more parallel. This orientation change was absent at sarcomere length 4.0 microm, where overlap between myosin and actin filaments is abolished. When the temperature of relaxed fibers was raised to 30 degrees C, the 6-ATR dipoles became more parallel to the fiber axis and less ordered; when ionic strength was lowered from 160 mM to 20 mM (5 degrees C), the 6-ATR dipoles became more perpendicular to the fiber axis and more ordered. In active contraction (10 degrees C), the orientational distribution of the probe dipoles was similar but not identical to that in relaxation, and was not a linear combination of the orientational distributions in relaxation and rigor.

Photolabile donors of nitric oxide: Ruthenium nitrosyl chlorides as caged nitric oxide

Abstract RuNOCl3 and K2RuNOCl5 are water-soluble, ionic caged NO compounds. On irradiation in the range 300–350 nm they liberate NO rapidly at a rate greater than 105 sec−1, with product quantum yields, Qp, of 0.012 and 0.06, respectively. The NO released on photolysis can be measured from an absorbance change associated with conversion of deoxyHb (FeII) to nitrosylHb (FeII). An alternative assay is to measure the absorbance change associated with conversion of oxyHb (FeIII) to metHb (FeIII) by photoreleased NO. When NO gas from a cylinder is dissolved in distilled water, the resulting solution also contains nitrous acid, which can be identified by its characteristic near-UV spectrum. A commonly used assay for NO dissolved in aqueous solvents involves diazotization of sulfanilic acid. This diazotization arises because of the action of the contaminating nitrite, not NO. Finally, the efficacy of the caged NO compounds in biological systems is shown by the relaxation of contracted aortic rings by photoreleased NO, just as occurs with endothelium-derived relaxing factor, which is known to be NO.