A Kv channel with an altered activation gate sequence displays both "fast" and "slow" activation kinetics

The Kv1-4 families of K+ channels contain a tandem proline motif (PXP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial "fast" mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered PXP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the PXP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the PXP motif may serve as a "hinge" and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them "reluctant" to open subsequently.     

Exponential decay kinetics in "downhill" protein folding

The observation of single-exponential kinetic phases in early stages of protein folding is often interpreted as evidence that these phases are rate limited by significant energy or entropy barriers. However, although the existence of large barriers reliably implies exponential kinetics, the reverse is not necessarily true. A simple model for the hydrophobic collapse of a chain molecule demonstrates that a barrierless or "downhill" diffusional relaxation can give rise to kinetics that are practically indistinguishable from a pure exponential. Within this model, even a highly nonlinear experimental probe such as resonance energy transfer (F?rster transfer) could exhibit a large amplitude decay (greater than 90% in fluorescence) that deviates from a simple exponential by less than 0.5%. Only a detailed analysis of the dynamics is likely to reveal that a free energy barrier is absent.     

A model of DSB-repair kinetics

A model of repair kinetics of DSB based on a step-by-step removal of individual double-strand breaks inside an autonomous repair unit is proposed and compared with the alternative model commonly used in modelling repair kinetics, which is based on an assumption of stochastic removal of cellular lesions. The former model seems to be preferred by the recently published experimental data displaying the time decrease of average DSB number per cell in irradiated Saccharomyces cerevisiae (strain 211 B) held in non-growth conditions.     

"The derivative assay"--an analysis of two fast components of DNA rejoining kinetics

The DNA rejoining kinetics of human U-118 MG cells were studied after gamma-irradiation with 4 Gy. The analysis of the sealing rate of the induced DNA strand breaks was made with a modification of the DNA unwinding technique. The modification meant that rather than just monitoring the number of existing breaks at each time of analysis, the velocity, at which the rejoining process proceeded, was determined. Two apparent first-order components of single-strand break repair could be identified during the 25 min of analysis. The half-times for the two components were 1.9 and 16 min, respectively.     

"Entropic traps" in the kinetics of phase separation in multicomponent membranes stabilize nanodomains

We quantitatively describe the creation and evolution of phase-separated domains in a multicomponent lipid bilayer membrane. The early stages, termed the nucleation stage and the independent growth stage, are extremely rapid (characteristic times are submillisecond and millisecond, respectively) and the system consists of nanodomains of average radius approximately 5-50 nm. Next, mobility of domains becomes consequential; domain merger and fission become the dominant mechanisms of matter exchange, and line tension gamma is the main determinant of the domain size distribution at any point in time. For sufficiently small gamma, the decrease in the entropy term that results from domain merger is larger than the decrease in boundary energy, and only nanodomains are present. For large gamma, the decrease in boundary energy dominates the unfavorable entropy of merger, and merger leads to rapid enlargement of nanodomains to radii of micrometer scale. At intermediate line tensions and within finite times, nanodomains can remain dispersed and coexist with a new global phase. The theoretical critical value of line tension needed to rapidly form large rafts is in accord with the experimental estimate from the curvatures of budding domains in giant unilamellar vesicles.     

A dose-response model incorporating nonlinear kinetics

This paper introduces a dose-response model for toxic quantal response data based on hit theory applied to the dose unit as transformed by a nonlinear kinetic equation. When spontaneous background response is included in the model, the resulting dose-response model has four parameters. The maximum likelihood estimators and their large-sample properties are given. Likelihood ratio tests of interest are developed, including one for whether the model is one-hit in the transformed dose and one to check whether nonlinear kinetics is operative. The use of the model for low-dose extrapolation is presented. Finally, the procedures developed are illustrated on data from three animal carcinogenicity bioassays that show, respectively, concave, linear, and convex dose-response curves in the observed data.     

Comparative kinetics of the sulphatases A

The kinetic behaviour of the sulphatase A from kangaroo liver is that of a simple hysteretic system involving a substrate-modified form of the enzyme. The equilibrium between the native and substrate-modified forms is influenced by one of the reaction products, sulphate. The behaviour of the system differs markedly from that involving the ox enzyme and a generalised model is presented to account for the behaviour of the sulphatases A from ox, human, rat and kangaroo livers.     

A structured model of dual-limitation kinetics

A structured model of substrate-utilization kinetics that encompasses dual-limitation conditions, caused by simultaneously low concentrations of the electron donor and the electron acceptor, is developed by incorporating the internal cofactor responses into the kinetic variables. The structured model is based on an assumption that the maximum specific electron-donor-oxidation rate (q(md)) is not a constant, but is linearly controlled by the intracellular chemical potentials, log(NAD/NADH) and log(ATP/ADP . P(i)). Determination of the kinetic parameters for the dual-limitation model, using experimental data from the companion article, verifies that q(md) varies and demonstrates that the NAD/NADH ratio affects q(md) in a positive direction; thus, an increase of the ratio increases the rate of electron-donor utilization. Because the internal NAD/NADH ratio rises with an increase in S(ar) the specific electron-donor-utilization rate is accelerated by high S(a). Since the ratio also increases as the specific electron-donor-utilization rate falls, the specific rate is intrinsically accelerated by the cofactor response when it becomes low due to a depletion of electron donor. Because the cofactor responses upon changes of the external substrate concentrations are systematic, the dual-limitation model can be expressed as a function of only external concentrations of electron donor and electron acceptor, which results in a multiplicative (double-Monod) form. Thus, dual limitation by both substrates reduces the overall reaction rate below the rate expected from single limitation by only one, the most severely limiting, substrate. (c) 1996 John Wiley & Sons, Inc.     

A light-scattering pressure-jump kinetics apparatus.

We have developed an optical sample cell made of stainless steel and fitted with three quartz ultracentrifuge windows in standard holders, to follow the kinetics of macromolecular reactions by the pressure-jump technique. Photomultiplier response to transmitted white light is continuously subtracted from photomultiplier response to white light scattered at 90°C, the difference being displayed by an oscilloscope. The pressure is simultaneously monitored by a quartz pressure sensor in mechanical contact with the sample. Pressurization is accomplished by leading in gas from a commercial cylinder, as originally described by Ljunggren and Lamm, but the pressurization time has been reduced by a factor of 25, to 2 millisec, by valving off a fixed volume of helium and introducing it into the sample cell through a high-speed solenoid valve. Determinations may be repeated at will on a single sample, of total volume under 2 ml. This light-scattering pressure-jump apparatus has been used to observe the kinetics of a number of macromolecular interactions and to determine rate constants for the ribosome-subunit interaction of Escherichia coli.     

Exploring "one-shot" kinetics and small molecule analysis using the ProteOn XPR36 array biosensor

A ProteOn XPR36 parallel array biosensor was used to characterize the binding kinetics of a set of small molecule/enzyme interactions. Using one injection with the ProteOn's crisscrossing flow path system, we collected response data for six different concentrations of each analyte over six different target protein surfaces. This "one-shot" approach to kinetic analysis significantly improves throughput while generating high-quality data even for low-molecular-mass analytes. We found that the affinities determined for nine sulfonamide-based inhibitors of the enzyme carbonic anhydrase II were highly correlated with the values determined using isothermal titration calorimetry. We also measured the temperature dependence (from 15 to 35 degrees C) of the kinetics for four of the inhibitor/enzyme interactions. Our results illustrate the potential of this new parallel-processing biosensor to increase the speed of kinetic analysis in drug discovery and expand the applications of real-time protein interaction arrays.     

A theoretical description of nitrate uptake kinetics in marine phytoplankton based on bisubstrate kinetics

Using well known equations for bisubstrate enzyme kinetics, a model for nitrate uptake in marine phytoplankton is generated. The model attempts to include both extracellular nitrate concentrations and incident light intensities as substrates required by the cells to take up nitrate. An affinity constant, K_LN, is defined which relates nitrate uptake kinetics to light intensities and extracellular nitrate concentrations simultaneously.     

A chemical kinetics model for microtubule oscillations

A model for microtubule oscillations is presented based on a set of chemical reaction equations. The rate constants for these reactions are largely determined from experimental data. The plots of assembled tubulin and the phase diagram for assembly are compared with the experimental findings and are found to agree quite well. Copyright 1999 Academic Press.