Detachment Rate Constants Research Articles

H2-Helical Region of Cardiac Troponin T Contributes to Length-Dependent Regulation of Cardiac Contractile Activation

An alpha-helical region of cardiac troponin T, cH2(T), is centrally positioned in the core domain of the troponin complex (Takeda et al., Nature 2003 vol.424(6944):p35-41). cH2(T)-troponin I and cH2(T)-troponin C interactions have been illustrated through biochemical studies and visualized in the crystal structure of the core domain of human cardiac troponin, suggesting an important regulatory role for cH2(T). However, little is known about how the cardiac-specific structure of cH2(T) relates to cardiac-specific contractile function. To better understand the functional significance of cH2(T), we created a chimeric rat cardiac troponin T (TnT) in which cH2(T) was replaced by the corresponding helical region of rat slow skeletal TnT and studied how replacement of native TnT with chimeric TnT affected contractile function of rat cardiac muscle. We measured isometric force, ATPase activity, and length-dependent contractile dynamics in detergent skinned papillary muscle bundles reconstituted with either wild-type or chimeric TnT, held at either sarcomere length (SL) 2.0 μm or 2.2 μm. Preliminary studies suggest that the SL dependence of Ca2+-activated maximal force production and tension cost was depressed in bundles containing chimeric TnT. For example, bundles containing the chimeric TnT showed an 8.4% decrease in force production when SL was decreased from 2.2 μm to 2.0 μm, whereas bundles containing the wild-type TnT showed a 43.9% decrease in force production. In addition, length-dependent contractile dynamics were significantly altered in bundles containing the chimeric TnT. For example, the length-dependent rate constant of crossbridge recruitment was slower, and the rate constant of crossbridge detachment was faster in bundles containing the chimeric TnT. Thus, our data suggest that cH2(T) plays a role in cardiac-specific length-mediated myofilament activation, an important mechanism underlying the Frank-Starling relationship.

Regulation of Contraction by PKA Phosphorylation of Myosin Binding Protein C and Troponin I in Murine Skinned Myocardium

In skinned myocardium, cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of cardiac myosin binding protein-C (cMyBP-C) and troponin I (cTnI) leads to a decrease in myofilament Ca2+-sensitivity and an acceleration in the kinetics of cross-bridge cycling. To examine the relative roles of cTnI and cMyBP-C phosphorylation in altering contractile function, we determined the Ca2+-sensitivity of force (pCa50) and the rate of force redevelopment (ktr) in untreated and PKA-treated murine myocardium expressing: (1) phosphorylatable cTnI and cMyBP-C (WT), (2) non-phosphorylatable cTnI with serines23/24/43/45 and threonine144 residues mutated to alanines (cTnIala5), (3) phosphorylatable cTnI on a cMyBP-C null background (cMyBP-C-/-), and (4) non-phosphorylatable cTnI on a cMyBP-C null background (cTnIala5/cMyBP-C-/-). A novel aspect of this study was the use of 2,3-Butandione Monoxime (BDM) treatments to reduce the basal levels of myosin regulatory light chain (RLC) phosphorylation to near zero in order to more accurately define the functional consequences of removing cMyBP-C and/or cTnI phoshorylation in transgenic myocardium. Our results showed that in the absence of RLC phosphorylation, PKA-treatment decreased pCa50 in WT, cTnIala5, and cMyBP-C-/- myocardium by 0.13, 0.08 and 0.09 pCa units, respectively, but had no effect in cTnIala5/cMyBP-C-/- myocardium. In WT and cTnIala5 myocardium, PKA treatment increased ktr at submaximal levels of activation; however, treatment did not have an effect on ktr in cMyBP-C-/- and cTnIala5/cMyBP-C-/- myocardium. Together, these results indicate that the attenuation of the myofilament force response following PKA treatment is due to phosphorylation of both cTnI and cMyBP-C and that the reduced Ca2+-sensitivity of force mediated by phosphorylation of cMyBP-C is most likely due to an increased rate constant of cross-bridge detachment that also contributes to an acceleration of cross-bridge cycling kinetics.

Low-energy electron attachment and detachment in vibrationally excited oxygen

Three-body electron attachment to O2 molecules and electron detachment from ions have been theoretically studied in vibrationally excited oxygen and O2-containing mixtures. Assuming that electron attachment and detachment proceed via the formation of vibrationally excited temporary ions, the rates of these processes were determined on the basis of the statistical approach for the vibrational transfer and relaxation in collisions between ions and O2 molecules. The calculated attachment and detachment rate constants turned out to agree well with available measurements in unexcited oxygen. This method was extended to calculate attachment and detachment rates in vibrationally excited oxygen. It was shown that the effect of vibrational excitation on electron detachment is profound, whereas attachment of low-energy electrons to vibrationally excited O2 is inefficient. The calculated vibrational distribution of stable ions turned out to be non-equilibrium in an excited gas and the effective vibrational temperature of the ions was much lower than the vibrational temperature of molecules. An analytical method was suggested to determine this distribution and the effective vibrational temperature. The calculated rate constants were used to simulate the formation and decay of an electron-beam-generated plasma in N2 : O2 mixtures at elevated vibrational temperatures. The calculations showed that vibrational excitation of molecules leads to orders of magnitude increase in the plasma density and in the plasma lifetime, in agreement with available observations.

On the accuracy of thermionic electron emission models. I. Electron detachment from SF6−

Detailed statistical rate calculations combined with electron capture theory and kinetic modeling for the electron attachment to SF(6) and detachment from SF(6)(-) [Troe et al., J. Chem. Phys. 127, 244303 (2007)] are used to test thermionic electron emission models. A new method to calculate the specific detachment rate constants k(det)(E) and the electron energy distributions f(E,epsilon) as functions of the total energy E of the anion and the energy epsilon of the emitted electrons is presented, which is computationally simple but neglects fine structures in the detailed k(det)(E). Reduced electron energy distributions f(E,epsilon/<epsilon>) were found to be of the form (epsilon/<epsilon>)(n) exp(-epsilon/<epsilon>) with n approximately = 0.15, whose shape corresponds to thermal distributions only to a limited extent. In contrast, the average energies <epsilon(E)> can be roughly estimated within thermionic emission and finite heat bath concepts. An effective temperature T(d)(E) is determined from the relation E - EA = <E(SF(6))(T(d))> + kT(d), where <E(SF(6))(T(d))> denotes the thermal internal energy of the detachment product SF(6) at the temperature T(d) and EA is the electron affinity of SF(6). The average electron energy is then approximately given by <epsilon(E)> = kT(d)(E), but dynamical details of the process are not accounted for by this approach. Simplified representations of k(det)(E) in terms of T(d)(E) from the literature are shown to lead to only semiquantitative agreement with the equally simple but more accurate calculations presented here. An effective "isokinetic" electron emission temperature T(e)(E) does not appear to be useful for the electron detachment system considered because it neither provides advantages over a representation of k(det)(E) as a function of T(d)(E), nor are recommended relations between T(e)(E) and T(d)(E) of sufficient accuracy.

Quantifying Nanoparticle Adhesion Mediated by Specific Molecular Interactions

Receptor-mediated targeting of nanometric contrast agents or drug carriers holds great potential for treating cardiovascular and vascular-associated diseases. However, predicting the ability of these vectors to adhere to diseased cells under dynamic conditions is complex due to the interplay of transport, hydrodynamic force, and multivalent bond formation dynamics. Therefore, we sought to determine the effects of adhesion molecule density and flow rate on adhesion of 210 nm particles, with the goal of identifying criteria to optimize binding efficiency and selectivity. Our system employed a physiologically relevant ligand, the vascular adhesion molecule ICAM-1, and an ICAM-1 specific antibody tethered to the nanoparticle using avidin-biotin chemistry. We measured binding and dissociation of these particles in a flow chamber as a function of antibody density, ligand density, and flow rate, and using a transport-reaction model we distilled overall kinetic rate constants for adhesion and detachment from the binding data. We demonstrate that both attachment and detachment of 210 nm particles can be correlated with receptor and ligand valency and are minimally affected by shear rate. Furthermore, we uncovered a time-dependent mechanism governing particle detachment, in which the rate of detachment decreases with contact time according to a power law. Finally, we use our results to illustrate how to engineer adhesion selectivity for specific molecular targeting applications. These results establish basic principles dictating nanoparticle adhesion and dissociation and can be used as a framework for the rational design of targeted nanoparticle therapeutics that possess optimum adhesive characteristics.

Pore-Scale Quantification of Colloid Transport in Saturated Porous Media

It is currently not clear how to quantifiably relate pore-scale observations of colloid transportto larger scales, so,we proposed a geometric theory showing that pore-scale-derived rate constants may be appropriate to model a larger scale system. This study considered three different types of colloids: latex microspheres, Escherichia coli, and microspheres made of poly lactic acid (PLA). Colloid attachment and detachment rate constants were calculated using digital microscope images, taken in rapid (1 s) sequences, from which rates of attaching and detaching colloids were readily observed. Average rate constants from >1000 images per colloid-type were used to model Darcy-scale colloid transport breakthrough curves. The modeled and observed breakthrough curves agreed well for all three types of colloids. However, for latex and PLA microspheres, the model systematically under predicted the breakthrough curves' rising limb, which may indicate that the rate "constants" are actually dependent on the amount of attached colloids. Insights into these sorts of complexities are best addressed by research that considers both pore-scale phenomena and larger-scale transport responses.

Quartz Crystal Microbalance Study of Protein Adsorption on Chitosan, Chitosan/Poly(vinyl pyrrolidone) Blends and Chitosan-graft-Poly(vinyl pyrrolidone) Surfaces

Adsorption behaviour of bovine serum albumin (BSA) onto chitosan (CS), chitosan/poly(vinyl pyrrolidone) blends (CS/PVP blends), and chitosan- graft-poly(vinyl pyrrolidone) (CS- graft-PVP) surfaces were investigated using quartz crystal microbalance (QCM). The adsorbed quantities of protein on the three surfaces were examined and were found to decrease in the following order: CS &gt; CS/PVP blends &gt; CS- graft-PVP. The kinetics of BSA adsorption in the early stage was analysed. The Langmuir isotherm model was used to describe the BSA adsorption behaviour on CS and its derivates. The attachment, detachment rate constants, and the association constants ( Ka ) were of the same order of magnitude, whereas the Ka for BSA on CS- graft-PVP surface was the lowest of the three surfaces. The adsorbed protein layer structure was also estimated. For CS- graft -PVP, most of the proteins adsorbed on the surface were of side-on structure, whereas for CS only about 40% of the proteins were of side-on structure.

Thin‐filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres

Thin-filament regulation of isometric force redevelopment (k(tr)) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I- and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca(2+)-activated force (rF(max)) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal k(tr) was unaffected until rF(max) was <0.2 of pre-extracted F(max). In contrast, reconstitution with cTnC or xsTnC reduced maximal k(tr) to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF(max) of 0.68 +/- 0.03 and 0.25 +/- 0.02 F(max). The k(tr)-pCa relation of fibres containing sTnC: xxsTnC mixtures (rF(max) > 0.2 F(max)) was little effected, though k(tr) was slightly elevated at low Ca(2+) activation. The magnitude of the Ca(2+)-dependent increase in k(tr) was greatly reduced following cTnC or xsTnC reconstitution because k(tr) at low levels of Ca(2+) was elevated and maximal k(tr) was reduced. Solution Ca(2+) dissociation rates (k(off)) from whole Tn complexes containing sTnC (26 +/- 0.1 s(-1)), cTnC (38 +/- 0.9 s(-1)) and xsTnC (50 +/- 1.2 s(-1)) correlated with k(tr) at low Ca(2+) levels and were inversely related to rF(max). At low Ca(2+) activation, k(tr) was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating k(tr) at any [Ca(2+)] and suggest Ca(2+) activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment.

Kinetics of colloidal alkylketene dimer particles deposition on pulp fibers

For achieving a certain level of hydrophobicity in paper, sizing agents are added to the paper stock. In alkaline papermaking system, sizing is often performed by the use of alkylketene dimer (AKD)-type sizing agents. Although the key factor for developing sizing properties in paper is the reaction between AKD and cellulose during drying, good retention of size particles in the paper web is required as a prerequisite for later reaction. A mathematical model based on particle collision frequency is used for the description of deposition kinetics of colloidal alkylketene dimer particles on pulp fibers. Modified Langmuir equation was used to describe the retention process in terms of surface coverage, attachment, and detachment rate constants. The values for equilibrium retention predicted by the model are in good agreement with experimental data. The analysis of surface charge in the system also suggests that the extent and efficiency of collisions between colloidal AKD size particles with fibers are one of the key factors for successful retention. Influence of shear on the retention process was also studied.

Dipole induced photodetachment dynamics of halide anions

In this article the dynamics of photodetachment from closed shell anion in the presence of two color (bichromatic) laser field have been explored in the context of polar environmental situation. The electronic states of halide ions are modeled by a one dimensional Hamiltonian with a potential V(x) = −V0e. The two parameters V0 and σ are fixed by requiring V(x) to reproduce the experimentally observed ground state ionization energy of the halide ion concerned. The potential so generated are shown to support only one bound state. The time-dependent Fourier grid Hamiltonian method is used to follow the detachment dynamics with fairly high intensities of light. The environmental effects on the dynamics are sought to be modeled by two ways i) allowing the well depth (V0) to fluctuate randomly V0(t) = V0[1+ΔVR(t)]; R(t) randomly fluctuates between +1 and −1 with time, when ΔV (strength of fluctuation) is fixed and ii) in the presence of perturbation produced by a neighboring solvent dipole, which changes the effective potential. The detachment rate constant is studied as a function of important system parameters of the used light field. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008

Pathway of ATP Hydrolysis by Monomeric Kinesin Eg5

Kinesin-5 family members including human Eg5/KSP contribute to the plus-end-directed force necessary for the assembly and maintenance of the bipolar mitotic spindle. We have used monomeric Eg5-367 in the nucleotide-free state to evaluate the role of microtubules at each step in the ATPase cycle. The pre-steady-state kinetic results show that the microtubule-Eg5 complex binds MgATP tightly, followed by rapid ATP hydrolysis with a subsequent slow step that limits steady-state turnover. We show that microtubules accelerate the kinetics of each step in the ATPase pathway, suggesting that microtubules amplify the nucleotide-dependent structural transitions required for force generation. The experimentally determined rate constants for phosphate product release and Eg5 detachment from the microtubule were similar, suggesting that these two steps are coupled with one occurring at the slow rate after ATP hydrolysis followed by the second step occurring more rapidly. The rate of this slow step correlates well with the steady-state k(cat), indicative that it is the rate-limiting step of the mechanism.

Kinetics of yeast detachment from controlled stainless steel surfaces

Using a radial flow chamber, we study Saccharomyces cerevisiae kinetics of detachment from stainless steel substrates. Samples of similar surface chemistry, but with different surface topologies are compared: mirror polished and electro-chemically etched. Different grain sizes (20, 40 and 100 μm) and different etching depths (100–650 nm) are tested. Cells are removed from the substrate according to a first-order kinetics defining two macroscopic parameters that depend on the applied stress: the detachment efficiency and the detachment rate constant. Whatever the surface topology, detachment occurs above a threshold and its rate is strongly stimulated by the applied stress. The detachment efficiency is characterized by the shear stress at which half of the cells detach and is independent of surface topology. In contrast, detachment is faster from etched than mirror polished surfaces. Finally, we also show the preferential adhesion of yeast cells to grains of <0 0 1> crystallographic orientation with respect to the surface.

DESIGN AND FUNCTION OF SUPERFAST MUSCLES: New Insights into the Physiology of Skeletal Muscle

Superfast muscles of vertebrates power sound production. The fastest, the swimbladder muscle of toadfish, generates mechanical power at frequencies in excess of 200 Hz. To operate at these frequencies, the speed of relaxation has had to increase approximately 50-fold. This increase is accomplished by modifications of three kinetic traits: (a) a fast calcium transient due to extremely high concentration of sarcoplasmic reticulum (SR)-Ca2+ pumps and parvalbumin, (b) fast off-rate of Ca2+ from troponin C due to an alteration in troponin, and (c) fast cross-bridge detachment rate constant (g, 50 times faster than that in rabbit fast-twitch muscle) due to an alteration in myosin. Although these three modifications permit swimbladder muscle to generate mechanical work at high frequencies (where locomotor muscles cannot), it comes with a cost: The high g causes a large reduction in attached force-generating cross-bridges, making the swimbladder incapable of powering low-frequency locomotory movements. Hence the locomotory and sound-producing muscles have mutually exclusive designs.

Increase in tension-dependent ATP consumption induced by cardiac troponin T mutation

How different mutations in cardiac troponin T (cTnT) lead to distinct secondary downstream cellular remodeling in familial hypertrophic cardiomyopathy (FHC) remains elusive. To explore the molecular basis for the distinct impact of different mutations in cTnT on cardiac myocytes, we studied mechanical activity of detergent-skinned muscle fiber bundles from different lines of transgenic (TG) mouse hearts that express wild-type cTnT (WTTG), R92W cTnT, R92L cTnT, and Delta-160 cTnT (deletion of amino acid 160). The amount of mutant cTnT is approximately 50% of the total myocellular cTnT in both R92W and R92L TG mouse hearts and approximately 35% in Delta-160 TG mouse hearts. Myofilament Ca2+ sensitivity was enhanced in all mutant cTnT TG cardiac muscle fibers. Compared with the WTTG fibers, Ca2+ sensitivity increased significantly at short sarcomere length (SL) of 1.9 microm (P < 0.001) in R92W TG fibers by 2.2-fold, in R92L by 2.0-fold, and in Delta-160 by 1.3-fold. At long SL of 2.3 microm, Ca2+ sensitivity increased significantly (P < 0.01) in a similar manner (R92W, 2.5-fold; R92L, 1.9-fold; Delta-160, 1.3-fold). Ca2+-activated maximal tension remained unaltered in all TG muscle fibers. However, tension-dependent ATP consumption increased significantly in Delta-160 TG muscle fibers at both short SL (23%, P < 0.005) and long SL (37%, P < 0.0001), suggesting a mutation-induced change in cross-bridge detachment rate constant. Chronic stresses on relative cellular ATP level in cardiac myocytes may cause a strain on energy-dependent Ca2+ homeostatic mechanisms. This may result in pathological remodeling that we observed in Delta-160 TG cardiac myocytes where the ratio of sarco(endo)plasmic reticulum Ca2+-ATPase 2/phospholamban decreased significantly. Our results suggest that different types of stresses imposed on cardiac myocytes would trigger distinct cellular signaling, which leads to remodeling that may be unique to some mutants.

Electron attachment and detachment:C6F5Cl,C6F5Br, andC6F5Iand the electron affinity ofC6F5Cl

Measurements are reported of rate constants for electron attachment to ${\mathrm{C}}_{6}{\mathrm{F}}_{5}X$ $(X=\mathrm{Cl},\mathrm{Br},\mathrm{I})$ and thermal electron detachment from ${\mathrm{C}}_{6}{\mathrm{F}}_{5}{\mathrm{Cl}}^{\ensuremath{-}}$ over the temperature range $300--550\phantom{\rule{0.3em}{0ex}}\mathrm{K}$ in $133\phantom{\rule{0.3em}{0ex}}\mathrm{Pa}$ of He gas in a flowing-afterglow Langmuir-probe apparatus. This is the first case we know of where the parent anion has sufficiently low electron detachment energy that detachment (from ${\mathrm{C}}_{6}{\mathrm{F}}_{5}{\mathrm{Cl}}^{\ensuremath{-}}$ in this case) has been observed in competition with a channel for dissociative electron attachment yielding a thermally stable anion (here, ${\mathrm{Cl}}^{\ensuremath{-}}$). Because of this competition, it is shown that a simple mass spectrometric determination of the product branching fractions at long times will lead to erroneous results at elevated temperatures. The electron density profiles provide evidence for a new plasma decay process involving the detaching and nondetaching anions trapped in the space charge field of the positive ions. Electron attachment rate constants were found to be $1.0\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}7}$, $1.1\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}7}$, and $2.0\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}7}\phantom{\rule{0.3em}{0ex}}{\mathrm{cm}}^{3}\phantom{\rule{0.2em}{0ex}}{\mathrm{s}}^{\ensuremath{-}1}$, at $300\phantom{\rule{0.3em}{0ex}}\mathrm{K}$, for ${\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{Cl}$, ${\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{Br}$, and ${\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{I}$, respectively, estimated accurate to $\ifmmode\pm\else\textpm\fi{}25%$ except for ${\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{I}$, where there is $\ifmmode\pm\else\textpm\fi{}30%$ uncertainty. Rate constants for ${\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{Cl}$ changed little over our temperature range, while those for ${\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{Br}$, and ${\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{I}$ increased with temperature. Electron detachment occurred only for ${\mathrm{C}}_{6}{\mathrm{F}}_{5}{\mathrm{Cl}}^{\ensuremath{-}}$ in our temperature range. Detachment rate constants were immeasurable at room temperature but approached $4000\phantom{\rule{0.3em}{0ex}}{\mathrm{s}}^{\ensuremath{-}1}$ at $550\phantom{\rule{0.3em}{0ex}}\mathrm{K}$. From these data the electron affinity (EA) for ${\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{Cl}$ was determined, EA $({\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{Cl})=0.75\ifmmode\pm\else\textpm\fi{}0.08\phantom{\rule{0.3em}{0ex}}\mathrm{eV}$. G3(MP2) calculations (based on M\o{}ller-Plesset perturbation theory) were carried out for the neutral and anion and yielded $\mathrm{EA}({\mathrm{C}}_{6}{\mathrm{F}}_{5}\mathrm{Cl})=0.728\phantom{\rule{0.3em}{0ex}}\mathrm{eV}$.

Photodetachment dynamics from closed‐shell anions in the presence of a bichromatic field

AbstractI have studied the dynamics of photodetachment from closed‐shell anions in the presence of a two‐color (bichromatic) laser field. The electronic states of halide ions are modeled by a 1‐D Hamiltonian with a potential V(x) = −V0e. The two parameters V0 and σ are fixed by requiring V(x) to reproduce the experimentally observed ground‐state ionization energy of the halide ions concerned. The potentials so generated are shown to support only one bound state in each case. The time‐dependent Fourier grid Hamiltonian method is used to follow the detachment dynamics with fairly high intensities of light. The environmental effects on the dynamics are sought to be modeled by allowing the well depth (V0) to fluctuate randomly (V0(t) = V0[1 + ΔVR(t)]; R(t) randomly fluctuates between +1 and −1 with time, when ΔV is fixed). The average detachment rate constants kav are seen to increase with increase in the intensities of used bichromatic field. An alternative model potential, V(x) = −V0e−σx, is also shown to yield similar results. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005

The Spatial and Temporal Dynamics of Pleckstrin Homology Domain Binding at the Plasma Membrane Measured by Imaging Single Molecules in Live Mouse Myoblasts

Pleckstrin homology (PH) domains act to target proteins to the plasma membrane and intracellular vesicles by binding to specific phosphoinositol phospholipids. We have investigated the binding kinetics of PH domains found in the tail region of the molecular motor, myosin X. Using total internal reflection fluorescence microscopy, we observed binding and release of individual PH domains fused to green fluorescent protein at the plasma membrane of living cells. Individual spots of light corresponding to single fluorescently tagged molecules were imaged onto a sensitive camera system, and digital image processing was then used to identify each fluorophore and store its trajectory in time and space. The PH domains bound with an apparent on-rate of 0.03 microm(-1) microm(-2) s(-1) and a detachment rate constant of 0.05 s(-1). The average residency time of the domains at the plasma membrane was about 20s. We found very limited movement of the membrane-bound PH domains in the mouse myoblast cells that we studied. This implies that the PH domains must either be attached to the cytoskeleton or corralled in a lipid compartment. Localization of the PH domains together with their rapid detachment rate is probably important in controlling the response of myosin X to signaling events and in regulating its cellular function.

Electron attachment and detachment and the electron affinity of cyclo-C4F8.

New measurements have been made of rate constants for electron attachment to c-C(4)F(8) (octafluorocyclobutane) and thermal electron detachment from the parent anion, c-C(4)F(8) (-), over the temperature range 298-400 K in 133 Pa of He gas in a flowing-afterglow Langmuir-probe apparatus. From these data the electron affinity for c-C(4)F(8) was determined, EA(c-C(4)F(8))=0.63+/-0.05 eV. The motivation was to resolve a discrepancy between our earlier EA estimate and a higher value (EA=1.05+/-0.10 eV) reported from a recent experiment of Hiraoka et al. [J. Chem. Phys. 116, 7574 (2002)]. The electron attachment rate constant is 9.3+/-3.0x10(-9) cm(3) s(-1) at 298 K. The electron detachment rate constant is negligible at room temperature but climbs to 1945+/-680 s(-1) at 400 K. G3(MP2) calculations were carried out for the neutral (D(2d), (1)A(1)) and anion (D(4h), (2)A(2u)) and yielded EA(c-C(4)F(8) (-))=0.595 eV. Bond energies were also calculated for loss of F from c-C(4)F(8) and loss of F or F(-) from c-C(4)F(8) (-). From these, dissociative electron attachment is found to be endothermic by at least 1.55 eV.

Myo1c is designed for the adaptation response in the inner ear.

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADP-release mechanism, which allows it to adapt to mechanical load.

Electron Attachment and Detachment: Cyclooctatetraene

Electron attachment and detachment rate constants were measured for 1,3,5,7-cyclooctatetraene (COT) in a He/Ar buffer gas over the temperature range 295−365 K. A flowing afterglow Langmuir probe apparatus was used for this work. Within experimental uncertainty, the electron attachment rate coefficient is independent of temperature in this range, at a value of 3.5 ± 1.0 ×10-9 cm3 s-1. The electron detachment rate is negligible at room temperature, but climbs to 995 s-1 at 365 K. The attachment/detachment equilibrium constant implies that the electron affinity of COT is 0.57 ± 0.03 eV, in agreement with other studies using different methods. A vertical detachment transition from the planar anion leaves COT in a planar transition state for ring inversion. G2(MP2) and density functional calculations were carried out for COT neutral, anion, dianion, and the ring inversion transition state. The G2(MP2) ring inversion barrier height compares well with the experimental result from NMR studies.