Control of a redox reaction on lipid bilayer surfaces by membrane dipole potential

Nitro-2,1,3-benzoxadiazol-4-yl (NBD) group is a widely used, environment-sensitive fluorescent probe. The negatively charged dithionite rapidly reduces the accessible NBD-labeled lipids in liposomes to their corresponding nonfluorescent derivatives. In this study both the phospholipid headgroup and acyl chain NBD-labeled L-alpha-1,2-dipalmitoyl-sn-glycero-3-phospho-[N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolamine] (DPPN) and 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC), respectively, were employed. The correlation of both the rate coefficient k(1) of the redox reaction and the fluorescence properties of the two probes with the membrane dipole potential Psi in fluid dipalmitoylglycerophosphocholine (DPPC) liposomes is demonstrated. When Psi of the bilayer was varied (decreased by phloretin or increased by 6-ketocholestanol), the value for k1 decreased for both DPPN and NBD-PC with increasing Psi. For both fluorophores a positive correlation to Psi was evident for the relative fluorescence emission intensity (RFI, normalized to the emission of the fluorophore in a DPPC matrix). The relative changes in emission intensity as a function of Psi were approximately equal for both NBD derivatives. Changes similar to those caused by phloretin were seen when dihexadecylglycerophosphocholine (DHPC) was added to DPPC liposomes, in keeping with the lower dipole potential for the former lipid compound compared with DPPC. These effects of Psi on NBD fluorescence should be taken into account when interpreting data acquired using NBD-labeled lipids as fluorescent probes.     

Reliable protein folding on complex energy landscapes: the free energy reaction path

A theoretical framework is developed to study the dynamics of protein folding. The key insight is that the search for the native protein conformation is influenced by the rate r at which external parameters, such as temperature, chemical denaturant, or pH, are adjusted to induce folding. A theory based on this insight predicts that 1), proteins with complex energy landscapes can fold reliably to their native state; 2), reliable folding can occur as an equilibrium or out-of-equilibrium process; and 3), reliable folding only occurs when the rate r is below a limiting value, which can be calculated from measurements of the free energy. We test these predictions against numerical simulations of model proteins with a single energy scale.     

Variation on reaction norm in lodgepole pine natural populations

Variations on the norm of reaction among ten natural lodgepole pine populations sampled from three lodgepole pine subspecies (Pinus contorta ssp. contorta, ssp. latifolia and ssp. murrayana) were studied by using 20 year heights measured in 57 provenance test sites across interior British Columbia (B.C.). There were significant population by site interactions. Concurrent joint regression and the AMMI model were used to dissect these population by environmental interactions. Joint regression analysis indicated that three populations (from the northwest) had a negative linear regression coefficient with environmental deviation, three (from central and southeast sites) had a positive regression coefficient and four (from the southwest) had a zero regression coefficient. The AMMI model revealed a similar pattern of reaction norm among the ten populations. But the three significant IPCA axes, which captured twice as much of the G × E sum of squares than joint regression, were more effective in separating the ten populations and associating their performance with the climate of test sites and their origin. The variation patterns of reaction norm in lodgepole pine populations demonstrated that adaptation of lodgepole pine natural populations to the various physical environments, at sub-species as well as at population level, was due largely to a balance between selection for high growth potential in less severe environments and selection for high cold hardiness in severe environments. Received: 4 May 2000 / Accepted: 10 November 2000     

Rotating horizontal ground reaction forces to the body path of progression

When studying the biomechanics of a transient turn, the orientation of the body will change relative to the orientation of the force plates over the progression of the turn. To express ground reaction forces relative to the body, this study investigated possible origin locations and axis alignments of body reference frames. The gait patterns of 10 subjects were recorded as subjects negotiated a 90 degrees hallway corner. Body reference frames were chosen whose origins were the center of mass (COM) and the pelvis origin (PEL). A finite-difference method was used to align the axes of the reference frames according to the horizontal paths of the COM and PEL. The ground reaction impulses (GRIs) were calculated relative to the COM and PEL reference frames. GRI differences were small between the PEL and COM frames, suggesting that either is acceptable for turning studies. Based on an investigation of finite-difference parameters, the COM frame should be used when using a kinematic sampling rate of 60 Hz. Either frame is acceptable when sampling at higher rates.     

On the role of polarizability in QSAR

The polarizability of a molecule, an important physical property, is currently attracting our attention particularly in the area of QSAR for chemical-biological interactions. In this report, the polarizability effects on ligand-substrate interactions has been discussed in terms of NVE (number of valence electrons) using additive values for valence electrons and the formulation of a total number of 51 QSAR. The QSAR model can be illustrated by Eq. I. log 1/C = a(NVE) +/- constant     

Variation of the oxidation state of verdoheme in the heme oxygenase reaction

Heme oxygenase (HO) converts hemin to biliverdin, CO, and iron applying molecular oxygen and electrons. During successive HO reactions, two intermediates, α-hydroxyhemin and verdoheme, have been generated. Here, oxidation state of the verdoheme-HO complexes is controversial. To clarify this, the heme conversion by soybean and rat HO isoform-1 (GmHO-1 and rHO-1, respectively) was compared both under physiological conditions, with oxygen and NADPH coupled with ferredoxin reductase/ferredoxin for GmHO-1 or with cytochrome P450 reductase for rHO-1, and under a non-physiological condition with hydrogen peroxide. EPR measurements on the hemin-GmHO-1 reaction with oxygen detected a low-spin ferric intermediate, which was undetectable in the rHO-1 reaction, suggesting the verdoheme in the six-coordinate ferric state in GmHO-1. Optical absorption measurements on this reaction indicated that the heme degradation was extremely retarded at verdoheme though this reaction was not inhibited under high-CO concentrations, unlike the rHO-1 reaction. On the contrary, the Gm and rHO-1 reactions with hydrogen peroxide both provided ferric low-spin intermediates though their yields were different. The optical absorption spectra suggested that the ferric and ferrous verdoheme coexisted in reaction mixtures and were slowly converted to the ferric biliverdin complex. Consequently, in the physiological oxygen reactions, the verdoheme is found to be stabilized in the ferric state in GmHO-1 probably guided by protein distal residues and in the ferrous state in rHO-1, whereas in the hydrogen peroxide reactions, hydrogen peroxide or hydroxide coordination stabilizes the ferric state of verdoheme in both HOs.     

Reversible folding of cysteine-rich metallothionein by an overcritical reaction path

A first-order-like state transition is considered to be involved in the restoration of the activities of a few proteins by correctly folding the protein [Phys. Rev. E 66 (2002) 021903]. In order to understand the general applicability of this mechanism, we studied a metallothionein (MT) protein with an unconventional structure, i.e., without any alpha-helix or beta-sheet. MT is a 61 amino-acid peptide. There are 6-7 Zn(2+) ions, which bind avidly to 20 conserved cysteines (Cys) of MT. These properties indicate that the structure of MT is quite different from those of the other proteins. Similar to our previous findings, the denatured MT can be folded without any aggregation via a designated stepwise quasi-static process (an over-critical reaction path). The particle size of folded MT intermediates, determined by dynamic light scattering, shrank right after the first folding stage. It is consistent with a collapse-model. In addition, results from both atomic absorption and circular dichroism (CD) indicate that the stable intermediates may fold to the native conformation but with only partial Zn(2+) binding, which in turn implies that those folding intermediates are in a molten globular state. These reversible unfolding and folding processes indicate that Cys-rich protein, MT, may also be folded by way of a first-order-like state transition mechanism. We suspect that this process may likely be involved in the reaction of the metal substitution process in metal containing enzymes.     

The mechanism of methanol decomposition by CuO. A theoretical study based on the reaction force and reaction electronic flux analysis

A theoretical study of methanol decomposition using a model representing the initial step of the reaction CH ₃ OH + CuO → CH ₂ O + H ₂ O + Cu is presented. Theoretical calculations using B3LYP/6-31 G along with Lanl2DZ pseudopotentials on metallic centers were performed and the results discussed within the framework of the reaction force analysis. It has been found that the reaction takes place following a stepwise mechanism: first, copper reduction (Cu ⁺² → Cu ⁺) accompanies the oxygen transposition and then a second reduction takes place (Cu ⁺ → Cu ⁰) together with a proton transfer that produce formaldehyde and release a water molecule.     

Effect of a molecular dipole on the ionic strength dependence of a biomolecular rate constant. Identification of the site of reaction.

A theory is proposed for determining the location of a reaction site on a protein of known tertiary structure with an asymmetric charge distribution by an analysis of the effect of ionic strength on the rate of reaction of the protein with a small ion, using equations of Brønsted (J. N. Brønsted, 1922, Z. Phys, Chem. 102:169-207), Debye and Hückel (P. Debye and E. Hückel, 1923, Phys. Z. 24:185-206), and Kirkwood (J. G. Kirkwood, 1934, J. Chem. Phys. 2:351-361). The theory is based on the fact that the dipole moment of the transition complex differs from that of the protein, which will be reflected in the ionic strength dependence of the reaction. The location of the small ion with respect to the dipole axis of the protein can be calculated from this difference. For protein-protein reactions, an a priori assumption has to be made about the orientation of one of the reaction partners, since many different orientations of the reactants with respect to each other result in dipole moments of the same magnitude.     

Theoretical calculation of polarizability isotope effects

We propose a scheme to estimate hydrogen isotope effects on molecular polarizabilities. This approach combines the any-particle molecular orbital method, in which both electrons and H/D nuclei are described as quantum waves, with the auxiliary density perturbation theory, to calculate analytically the polarizability tensor. We assess the performance of method by calculating the polarizability isotope effect for 20 molecules. A good correlation between theoretical and experimental data is found. Further analysis of the results reveals that the change in the polarizability of a X-H bond upon deuteration decreases as the electronegativity of X increases. Our investigation also reveals that the molecular polarizability isotope effect presents an additive character. Therefore, it can be computed by counting the number of deuterated bonds in the molecule.     

Delayed fluorescence from the photosynthetic reaction center measured by electronic gating of the photomultiplier

The decay of the delayed fluorescence (920 nm) of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides R26 in the P(+)Q(A)(-) charge-separated state (P and Q(A) are the primary donor and quinone, respectively) has been monitored in a wide (100 ns to 100 ms) time range. The photomultiplier (Hamamatsu R3310-03) was protected from the intense prompt fluorescence by application of gating potential pulses (-280 V) to the first, third, and fifth dynodes during the laser pulse. The gain of the photomultiplier dropped transiently by a factor of 1 x 10(6). The delayed fluorescence showed a smooth but nonexponential decay from 100 ns to 1 ms that was explained by the relaxation of the average free energy between P* and P(+)Q(A)(-) changing from -580 to -910 meV. This relaxation is due to the slow protein response to charge separation and can be described by a Kohlrausch relaxation function with time constant of 65 micros and a stretching exponent of alpha = 0.45.     

Electric dichroism of DNA. Influence of the ionic environment on the electric polarizability

In order to test the diffuse ion atmosphere polarization model recently developed by us, the effects of ionic strength, titrating with Mg2+ and Co(NH3)3+6, and coion charge on the electric polarizability of short fragments of DNA are investigated. The results are consistent with the predictions of the theory and show that the diffuse ion atmosphere polarization contributes significantly to the overall orientation of DNA. At low ionic strengths, we attempt to separate the total dipole moment into two components: one that agrees well with the Debye-Hückel ion atmosphere calculations, while the other, presumably due to condensed counterion polarization, appears to be substantially independent of the ionic strength. At higher salt concentrations, however, a simple separation into dipole components is not possible, perhaps due to a significant coupling of ion flows between the diffuse atmosphere and the condensed counterion layer.     

Influence of ethanol on the high frequency electric polarizability of E. coli

The interface electric polarizability of bacteria (charge dependent (ChD) and Maxwell-Wagner (MW) polarizabilities) gives information about their electric charge, determined by the structure and functional state. It is well known that the polarizability could be changed significantly by adding some substances to the suspension, and can be measured using an electro-optical (EO) method. There are some literature data, according to which the adding of ethanol decreases the electric polarizability of the cells. However the reason for the change in this parameter is not clear, as well as which component (ChD and/or MW) of polarizability has the main contribution. Generally the present work shows that the effect of ethanol is connected to the change of the internal (cytoplasm) MW polarizability and is mainly caused by increasing the cell membrane permeability. This results in an ionic flow through the membrane, which velocity and direction depends on the relative values of the inner (cytoplasm) and the outer medium ionic strength.     

Steric and electronic effects of the alkyl group on the activation parameters of the water substitution reaction of some cobaloximes

The activation parameters for the water substitution reactions of some organocobaloximes with thiourea in aqueous solution at I = 1 M (NaNO₃) are reported and discussed. Scatter in an isokinetic plot indicates that more than one interaction mechanism is at play. The ΔH* values are shown to be influenced by the electron withdrawing power and by the steric bulk of alkyl group. The influence of these factors on ΔS* cannot be rationalized.     

Enzymatic circularization of a malto-octaose linear chain studied by stochastic reaction path calculations on cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) is an enzyme belonging to the alpha-amylase family that forms cyclodextrins (circularly linked oligosaccharides) from starch. X-ray work has indicated that this cyclization reaction of CGTase involves a 23-A movement of the nonreducing end of a linear malto-oligosaccharide from a remote binding position into the enzyme acceptor site. We have studied the dynamics of this sugar chain circularization through reaction path calculations. We used the new method of the stochastic path, which is based on path integral theory, to compute an approximate molecular dynamics trajectory of the large (75-kDa) CGTase from Bacillus circulans strain 251 on a millisecond time scale. The result was checked for consistency with site-directed mutagenesis data. The combined data show how aromatic residues and a hydrophobic cavity at the surface of CGTase actively catalyze the sugar chain movement. Therefore, by using approximate trajectories, reaction path calculations can give a unique insight into the dynamics of complex enzyme reactions.