The mitochondrial channel VDAC has a cation-selective open state

The mitochondrial channel VDAC is known to have two major classes of functional states, a large conductance "open" state that is anion selective, and lower conductance substates that are cation selective. The channel can reversibly switch between open and half-open states, with the latter predominant at increasing membrane voltages of either polarity. We report the presence of a new functional state of VDAC, a cation-selective state with conductance approximately equal to that of the canonical open state. This newly described state of VDAC can be reached from either the half-open cation-selective state or from the open anion-selective state. The latter transition implies that a mechanism exists for selectivity gating in VDAC that is separate from partial closure, which may be relevant to the physiological regulation of this channel and mitochondrial outer membrane permeability.     

The kinetic and physical basis of K(ATP) channel gating: toward a unified molecular understanding

K(ATP) channels can be formed from Kir6.2 subunits with or without SUR1. The open-state stability of K(ATP) channels can be increased or reduced by mutations throughout the Kir6.2 subunit, and is increased by application of PIP(2) to the cytoplasmic membrane. Increase of open-state stability is manifested as an increase in the channel open probability in the absence of ATP (Po(zero)) and a correlated decrease in sensitivity to inhibition by ATP. Single channel lifetime analyses were performed on wild-type and I154C mutant channels expressed with, and without, SUR1. Channel kinetics include a single, invariant, open duration; an invariant, brief, closed duration; and longer closed events consisting of a "mixture of exponentials," which are prolonged in ATP and shortened after PIP(2) treatment. The steady-state and kinetic data cannot be accounted for by assuming that ATP binds to the channel and causes a gate to close. Rather, we show that they can be explained by models that assume the following regarding the gating behavior: 1) the channel undergoes ATP-insensitive transitions from the open state to a short closed state (C(f)) and to a longer-lived closed state (C(0)); 2) the C(0) state is destabilized in the presence of SUR1; and 3) ATP can access this C(0) state, stabilizing it and thereby inhibiting macroscopic currents. The effect of PIP(2) and mutations that stabilize the open state is then to shift the equilibrium of the "critical transition" from the open state to the ATP-accessible C(0) state toward the O state, reducing accessibility of the C(0) state, and hence reducing ATP sensitivity.     

A study of dense mineralized tissue by neutron diffraction

Neutron diffraction studies of mineralized tissue show a close relationship between the wet state equatorial diffraction spacing and wet tissue density expressable as a second-order polynomial. The molecular fractional shrinkage when the tissue is dried shows a straight line dependence on wet tissue density with a correlation of 0.98. Since the dry state equatorial diffraction spacing is much less than for the corresponding wet state, even in fully mineralized bone, the collagen molecules must be displaced through a mineral-free volume while drying. The mineral can only be located within the available volume of the dried tissue whether intra- or extrafibrillar. The dimension of the dry state equatorial spacing for each of the tissues examined is close to that of dried tendon collagen. It appears unlikely that hydroxyapatite crystallites can be accommodated radially between collagen molecules in bone if the packing is like that of dried tail tendon collagen. The only mineral within the fibrils must be in the intermolecular gaps. It is estimated on the basis of the volume of the axial intermolecular gaps and the minimum extrafibrillar volume that the intrafibrillar mineral can be no more than 20% of the total mineral and may be less than 10%.     

The low-pH unfolded state of the C-terminal domain of the ribosomal protein L9 contains significant secondary structure in the absence of denaturant but is no more compact than the low-pH urea unfolded state

There is considerable interest in the properties of the unfolded states of proteins, particularly unfolded states which can be populated in the absence of high concentrations of denaturants. Interest in the unfolded state ensemble reflects the fact that it is the starting point for protein folding as well as the reference state for protein stability studies and can be the starting state for pathological aggregation. The unfolded state of the C-terminal domain (residues 58-149) of the ribosomal protein L9 (CTL9) can be populated in the absence of denaturant at low pH. CTL9 is a 92-residue globular alpha, beta protein. The low-pH unfolded state contains more secondary structure than the low-pH urea unfolded state, but it is not a molten globule. Backbone ( (1)H, (13)C, and (15)N) NMR assignments as well as side chain (13)C beta and (1)H beta assignments and (15)N R 2 values were obtained for the pH 2.0 unfolded form of CTL9 and for the urea unfolded state at pH 2.5. Analysis of the deviations of the chemical shifts from random coil values indicates that residues that comprise the two helices in the native state show a clear preference for adopting helical phi and psi angles in the pH 2.0 unfolded state. There is a less pronounced but nevertheless clear tendency for residues 107-124 to preferentially populate helical phi and psi values in the unfolded state. The urea unfolded state has no detectable tendency to populate any type of secondary structure even though it is as compact as the pH 2.0 unfolded state. Comparison of the two unfolded forms of CTL9 provides direct experimental evidence that states which differ significantly in their secondary structure can have identical hydrodynamic properties. This in turn demonstrates that global parameters such as R h or R g are very poor indicators of "random coil" behavior.     

Kinetics of activation and desensitization in receptor proteins

Receptors are functional membrane proteins on the cell surface that recognize external signals and trigger biological responses by generating intracellular signals. Due to prolonged exposure to external signals, receptors are often desensitized and no longer produce intracellular signals. This simple control mechanism may work without negative-feedback regulation from another molecule if the active state of a receptor reflects a transient metastable molecular structure. A theoretical framework is developed to identify a metastable state associated with a conformational transition of protein molecules, in which a transient state can be observed somewhat above the equilibrium transition point. The conducting state of the acetylcholine receptor may thus represent a metastable state associated with a conformational transition from the resting state to the desensitized state. Similarly, the conducting state of the voltage-sensitive sodium channel may represent a metastable state associated with a conformational transition from the resting state to the refractory state. The rates of appearance and disappearance of the transient state, as well as the equilibrium ratio of the two preexisting states, can be estimated from the free energy of protein structure. The appearance of the transient state is generally a multirelaxation process and may show a time lag, while the disappearance is a slower single-relaxation process.     

Physiological state-specific models in estimation of recombinant Escherichia coli fermentation performance

Implementation of advanced control strategies in bioprocesses is often hindered by the lack of on-line measurements reflecting the physiological state of the culture. Although a number of techniques have been used to estimate key variables from data monitored on-line, these often do not explicitly take into account changes in physiological state and information on many aspects of physiological state that may not be present in on-line data. Here we demonstrate that data obtained from chemical fingerprinting methods, such as pyrolysis mass spectrometry, can be used to identify changes in the physiological state during cultivation. This information can be utilized for the estimation of the physiological state and can enable physiological state-specific-model development for on-line bioprocess control.     

Optical ellipsometry on the diffraction order of skinned fibers. pH-induced rigor effects.

The polarization properties of light diffracted from single-skinned fibers of skeletal muscles have been examined under conditions in which the bathing solution pH and the ionic strength are changed. For fibers in the relaxed state, we observe large decreases in both the total depolarization signal, r, and the total diffraction birefringence signal, delta nT, upon pH change from 7.0 to 8.0 at normal ionic strength. However, if the ionic strength is raised, then the r-value change as the pH changes from pH 7.0 to pH 8.0 is much smaller. If the rigor state is achieved at pH 8.0, and 0 mM ATP under either of the ionic strength conditions, the fiber can still be stretched. Rigor stiffness for this state is only approximately 20% that of the value of the stiffness at pH 7.0 rigor. Electron micrographs obtained under this pH 8.0 rigor state show that the overlap region can be decreased upon stretching the fiber, signifying a different kind of weaker-binding rigor state. Optically, the weaker-binding rigor state has a lower depolarization signal and larger form birefringence than the strong-binding rigor state. To convert from one type of rigor state (pH 7.0) to the other rigor state (pH 8.0), or vice versa, the fiber must first be relaxed. Apparently, either of the rigor states can block the full impact of the pH effect.     

Determination of the protonation state of the Asp dyad: conventional molecular dynamics versus thermodynamic integration

The protonation state of the Asp dyad is important as it can reveal enzymatic mechanisms, and the information this provides can be used in the development of drugs for proteins such as memapsin 2 (BACE-1), HIV-1 protease, and rennin. Conventional molecular dynamics (MD) simulations have been successfully used to determine the preferred protonation state of the Asp dyad. In the present work, we demonstrate that the results obtained from conventional MD simulations can be greatly influenced by the particular force field applied or the values used for control parameters. In principle, free-energy changes between possible protonation states can be used to determine the protonation state. We show that protonation state prediction by the thermodynamic integration (TI) method is insensitive to force field version or to the cutoff for calculating nonbonded interactions (a control parameter). In the present study, the protonation state of the Asp dyad predicted by TI calculations was the same regardless of the force field and cutoff value applied. Contrary to the intuition that conventional MD is more efficient, our results clearly show that the TI method is actually more efficient and more reliable for determining the protonation state of the Asp dyad.     

Past states of continuous-time Markov models for ecological communities

Discrete-time Markov chains are often used to model communities of sessile organisms. The community is described by a set of discrete states, which may represent species or groups of species. Transitions between states are modelled using a stochastic matrix. A recent study showed how the time-reversal of such a Markov chain can be used to estimate the distribution of time since the last occurrence of some state of interest (such as empty space) at a point, given the current state of the point. However, if the underlying process operates in continuous time but is observed at regular intervals, this distribution describes the time since the last possible observation of the state of interest, rather than the time since its last occurrence. We show how to obtain the distribution of time since the last occurrence of a state of interest for a continuous-time homogeneous Markov chain. The expected time since the last occurrence of an initial state can be interpreted as a measure of the successional rank of a state. We show how to distinguish between different ways in which a state can have high successional rank. We apply our results to a marine subtidal community.     

Base stacking and unstacking as determined from a DNA decamer containing a fluorescent base

Time-resolved fluorescence decay of a single-stranded DNA decamer d(CTGAAT5CAG), where d5 is the fluorescent base 1-(beta-D-2'-deoxyribosyl)-5-methyl-2-pyrimidinone, was measured and analyzed at several temperatures. The d5 base in the decamer is resolved into three states according to their fluorescence decay lifetime characteristics and temperature dependence of their associated amplitudes: fully extended and completely unstacked state, loosely associated state, and fully stacked state. These states are in slow exchange compared to their fluorescence decay rates. The population of the fully extended and completely unstacked state is small and decreases further with increasing temperature. The loosely associated state, whose fluorescence can still be efficiently quenched by other DNA bases, occupies a large portion of the conventionally defined unstacked state. Stacking enthalpy and entropy for the d5 base with thymine or cytosine bases in the DNA decamer are calculated to be -6.6 kcal/mol and -22 cal/mol.K, respectively. This work shows that fluorescent bases in DNA can be useful to the study of local conformations of bases.     

Graphic rules in steady and non-steady state enzyme kinetics

Graphic methods, when applied to enzyme kinetics, can provide a visually intuitive relation between calculations and reaction graphs. This will not only greatly raise the efficiency of calculations but also significantly help the analysis of enzyme kinetic mechanisms. In this paper, four graphic rules are presented. Rules 1-3 are established for steady state enzyme-catalyzed reaction systems and Rule 4 is for non-steady state ones. In comparison with conventional graphic methods which can only be applied to steady state systems, the present rules have the following merits. 1) Complicated and tedious calculations can be greatly simplified; for example, in calculating the concentrations of enzyme species for the bi-bi random mechanism, the calculation work can be reduced 8-fold compared with the King-Altman's method. 2) A great deal of wasted labor can be avoided; for example, in calculating the rate of product formation for the same mechanism, the operation of finding and removing the 96 reciprocally canceled terms is no longer needed because they automatically disappear during the derivation. 3) Final results can be easily and safely checked by a formula provided in each of the graphic rules. 4) Non-steady state systems can also be treated by the present graphic method; for example, applying Rule 4, one can directly write out the solution for a non-steady state enzyme-catalyzed system, without the need to follow more difficult and complicated operations to solve differential equations. The mathematical proofs of Rules 1-4 are given in Appendices A-D (in the Miniprint), respectively.     

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.     

A Simple Self-Maintaining Metabolic System: Robustness,Autocatalysis, Bistability

A living organism must not only organize itself from within; it must also maintain its organization in the face of changes in its environment and degradation of its components. We show here that a simple (M,R)-system consisting of three interlocking catalytic cycles, with every catalyst produced by the system itself, can both establish a non-trivial steady state and maintain this despite continuous loss of the catalysts by irreversible degradation. As long as at least one catalyst is present at a sufficient concentration in the initial state, the others can be produced and maintained. The system shows bistability, because if the amount of catalyst in the initial state is insufficient to reach the non-trivial steady state the system collapses to a trivial steady state in which all fluxes are zero. It is also robust, because if one catalyst is catastrophically lost when the system is in steady state it can recreate the same state. There are three elementary flux modes, but none of them is an enzyme-maintaining mode, the entire network being necessary to maintain the two catalysts.     

Pitfalls of the 45Ca uptake method

The interpretation of the data derived from ⁴⁵Ca uptake measurements by cells and tissues can be difficult. Several of these difficulties and possible misinterpretations are described: 1) ⁴⁵Ca uptake is not equivalent to calcium influx; 2) interpretations based on the sole visual examination of ⁴⁵Ca uptake curves can be misleading because an increased tracer uptake can coexist with a decreased calcium transport and vice-versa; 3) drugs and hormones can have diametrically opposite effects when they are tested at steady state on in nonsteady state conditions. It is concluded that ⁴⁵Ca uptake curves must be kinetically analyzed and that the steady or non-steady state of the system must be known for a valid interpretation of such data.     

Biological models: measuring variability with classical and quantum information

This essay proposes methods to analyse the variability of biological data. The idea is to express the state of a biological system as a linear combination of base states in a Hilbert space. Coefficients of the linear combination can be interpreted as probabilities and informational entropy is associated to each state allowing the definition of a classical variability measure. Besides, state transition matrices can also be calculated and their norms express the dynamics of the system organization and a quantum variability measure. As the examples show, the classical measure expresses a structural variability and the quantum measure expresses a functional variability.     

Analysis of bifurcations in continuous fermentation using recombinant microorganisms with delayed responses

When the feed rate to a fermenter is varied periodically in order to favor the growth of plasmid-containing cells, a transition may occur from the starting stationary state to another state. The resulting state may be constant or oscillatory. A generalised model based on the adaption times of plasmid-free and plasmid-harboring cells has been used. Analytical conditions have been derived for bifurcation from one nonoscillatory state to another or to an oscillatory state (Hopf bifurcation). The frequency of oscillation is shown to have an upper bound, which can be controlled by manipulating certain process parameters. The production of tryptophan synthetase by the plasmid pPLc23trpAl in E. coli is used as an example to determine the nature of the Hopf bifurcations.     

On the mechanism of the chemical modification of the mitochondrial acetyl-CoA acetyltransferase by coenzyme A

The liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), is involved in ketone body synthesis. The enzyme can be chemically modified and inactivated by CoASH and also by CoASH-disulfides provided glutathione is present. The unmodified enzyme shows in its denatured state 7.95 +/- 0.44 sulfhydryl groups per enzyme and in its native state 3.92 +/- 0.34 sulfhydryl groups which react with Ellmann's reagent. The modified enzyme reveals in its native state also 4.07 +/- 0.25 sulfhydryl groups per enzyme, but in its denatured state 9.10 +/- 0.51 sulfhydryl groups could be detected. Approximately four sulfhydryl groups per enzyme, unmodified or modified, can be alkylated by iodoacetamide. These results prove for each subunit the existence of two sulfhydryl groups and suggest the existence of two disulfide bridges. The CoASH modification, which should proceed at one of these disulfide groups, prevents subsequent acetylation of the enzyme and is drastically reduced in the iodoacetamide-alkylated enzyme. In the demodification of the modified enzyme, the CoASH is set free as a mixed disulfide with glutathione.