John E. T. Corrie

Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20–40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a ‘hand-over-hand’ mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.

Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules

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 M1 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 ~30 ms and ~20 nm. In isolated CHO cells, receptors are randomly distributed over the plasma membrane. At any given time, ~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 M1 receptors undergoing interconversion between monomers and dimers on the timescale of seconds.

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.

Photochemical and pharmacological evaluation of 7-nitroindolinyl-and 4-methoxy-7-nitroindolinyl-amino acids as novel, fast caged neurotransmitters

Reagents capable of rapid and efficient release of neuroactive amino acids (L-glutamate, GABA and glycine) upon flash photolysis of thermally stable, inert precursors have been elusive. 7-Nitroindolinyl (NI)-caged and 4-methoxy-7-nitroindolinyl (MNI)-caged compounds that fulfil these criteria are evaluated here. These caged precursors are highly resistant to hydrolysis. Photolysis is fast (half time< or =0.26 ms) and the conversion achieved with a xenon flashlamp is about 15% for the NI-caged L-glutamate and about 35% for the MNI-caged L-glutamate. A procedure is described for calibration of photolysis in a microscope-based experimental apparatus. NI-caged L-glutamate itself showed no agonist or antagonist effects on AMPA and NMDA receptors in cultured neurones, and had no effect on climbing fibre activation of Purkinje neurones. A control compound with identical photochemistry that generated an inert phosphate upon photolysis was used to confirm that the intermediates and by-products of photolysis have no deleterious effects. MNI-caged L-glutamate is as stable and fast as NI-caged L-glutamate and similarly inert at glutamate receptors, but about 2.5 times more efficient. However, NI-caged GABA is an antagonist at GABA(A) receptors and NI-glycine an antagonist at glycine receptors. The results show the utility and limitations of these fast and stable caged neurotransmitters in the investigation of synaptic processes.

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.

Effects of Aromatic Substituents on the Photocleavage of 1-Acyl-7-nitroindolines

Abstract Photolysis of 1-acyl-7-nitroindolines in aqueous solution gives a carboxylic acid and a 7-nitrosoindole. These compounds are useful as photolabile precursors of carboxylic acids, particularly neuroactive amino acids. The effect of electron-donating substituents at the 4-position has been explored for its effect on photolysis efficiency. 4-Methoxy substitution improved the photolysis efficiency >2-fold but the 4-dimethylamino analogue was essentially inert. A 5-alkyl substituent, that blocks unwanted nitration at this position, reduced the beneficial effect of the 4-methoxy group.

Fluorescence Polarization Transients from Rhodamine Isomers on the Myosin Regulatory Light Chain in Skeletal Muscle Fibers

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.

The efficiency of contraction in rabbit skeletal muscle fibres, determined from the rate of release of inorganic phosphate

1 The relationship between mechanical power output and the rate of ATP hydrolysis was investigated in segments of permeabilized fibres isolated from rabbit psoas muscle. 2 Contractions were elicited at 12 °C by photolytic release of ATP from the P3‐1‐(2‐nitrophenyl)ethyl ester of ATP (NPE‐caged ATP). Inorganic phosphate (Pi) release was measured by a fluorescence method using a coumarin‐labelled phosphate binding protein. Force and sarcomere length were also monitored. 3 ATPase activity was determined from the rate of appearance of Pi during each phase of contraction. The ATPase rate was 10.3 s−1 immediately following release of ATP and 5.1 s−1 during the isometric phase prior to the applied shortening. It rose hyperbolically with shortening velocity, reaching 18.5 s−1 at a maximal shortening velocity > 1 ML s−1 (muscle lengths s−1). 4 Sarcomeres shortened at 0.09 ML s−1 immediately following the photolytic release of ATP and at 0.04 ML s−1 prior to the period of applied shortening. The high initial ATPase rate may be largely attributed to initial sarcomere shortening. 5 During shortening, maximal power output was 28 W l−1. Assuming the free energy of hydrolysis is 50 kJ mol−1, the efficiency of contraction was calculated from the power output at each shortening velocity. The maximum efficiency was 0.36 at a shortening velocity of 0.27 ML s−1, corresponding to a force level 51 % of that in the isometric state. 6 At the maximal shortening velocity, only 10 % of the myosin heads are attached to the thin filaments at any one time.

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.

The conductance underlying the parallel fibre slow EPSP in rat cerebellar Purkinje neurones studied with photolytic release of L‐glutamate

1 Tetanic stimulation of parallel fibres (PFs) produces a slow EPSP (sEPSP) or slow EPSC (sEPSC) in Purkinje neurones (PNs), mediated by type 1 metabotropic glutamate receptors (mGluR1). The conductance change underlying the sEPSP was investigated with rapid photolytic release of L‐glutamate from nitroindolinyl (NI)‐caged glutamate with ionotropic glutamate receptors blocked, and showed a slow mGluR1‐activated cation channel. 2 In cerebellar slices rapid photolytic release (t1/2 < 0.7 ms) of 7‐70 μM L‐glutamate on PNs voltage clamped at −65 mV activated first a transient inward current, peaking in 8 ms, followed by a slow inward current with time course similar to the PF sEPSP, peaking at −1 nA in 700 ms. 3 The initial current was inhibited by 300 μM threo‐hydroxyaspartate (THA) and did not reverse as the potential was made positive up to +50 mV, suggesting activation of electrogenic glutamate uptake. 4 The slow current was inhibited reversibly by 1 mM (R,S)‐MCPG or the non‐competitive mGluR1 antagonist CPCCOEt (20 μM), indicating activation of metabotropic type 1 glutamate receptors. The mGluR current was associated with increases of input conductance and membrane current noise, and reversed close to 0 mV, indicating activation of channels permeant to Na+ and K+. 5 The sEPSC was not blocked by Cd2+, Co2+, Mg2+ or Gd3+ ions, by the inhibitor of hyperpolarisation‐activated current (IH) ZD7288, or by the purinoceptor inhibitor PPADS. Activation was not affected by inhibitors of phospholipase C (PLC) or protein kinase C (PKC), nor mimicked by photorelease of InsP3 or Ca2+. The results show that mGluR1 in PNs produces a slow activation of cation‐permeable ion channels which is not mediated by PLC activation, Ca2+ release from stores, or via the activation of PKC.