Configuration-dependent properties of randomly coiling polynucleotide chains. II. The role of the phosphodiester linkage

The dependence of the unperturbed dimensions of randomly coiling polynucleotides on the rotations about the phosphodiester linkages of the chain has been examined in order to understand the conformational discrepancies, set forth in paper I, regarding these angles (ω′ and ω). Large values of the characteristic ratio 〈r²〉₀/nl² , which agree with the experimental behavior of the chain, are obtained only if a sizeable proportion of the polymer residues have trans ω′ values. The asymmetric torsional potential that is believed to arise from gauche effects associated with the P-O bonds has been approximated using a hard core model. The calculated characteristic ratio exhibits a strong dependence upon the magnitude of this torsional barrier (separating trans and gauche conformations) and shows agreement with experimental values for polyribonucleotides only if this energy difference is 1 kcal/mol or less.     

Magnesium-DNA interactions from interpretation of 25Mg-nmr relaxation rates: Field and coion dependence

We have used ²⁵Mg-nmr to investigate the binding of magnesium ions to double-stranded DNA. We have measured line shapes for ²⁵Mg in the presence of monodisperse calf thymus DNA (160 base pairs; b.p.) (magnesium : phosphate = 2.0) at two different field strengths, 11.75 T and 7.05 T, and used the isotropic model of two-site exchange developed by Westlund and Wennerstrom to simultaneously fit the line shapes at both field strengths. This model does not reproduce the observed field dependence. This is in contrast to a previous study [E. Berggren, L. Nordenskiold, and W. H. Braunlin (1992), Biopolymers, Vol. 32, pp. 1339–1350] in which a similar model of isotropic two-site exchange qualitatively reproduced the temperature dependence of the line widths. Relaxation rates were also measured as a function of magnesium : phosphate ratio and colon type. These measurements were used to assess the sensitivity of magnesium relaxation measurements to small changes in DNA structure induced by changes in the solvent environment. The temperature dependence of the line shape varies with the type of coion (chloride or sulfate) present. This coion dependence of the line shape is consistent with the coion dependence of the aggregation midpoint temperature reported by Bloomfield and co-workers [O. A. Knoll, M. G. Fried, and V. A. Bloomfield (1988) in Structure and Expres-sion, Vol. 2, R. H. Sarma and M. H. Sarma, Eds., Adenine Press, New York] and attributed to a lyotropic effect. These results suggest that even at low magnesium : phosphate ratios, relaxation parameters are specific to each magnesium–coion–DNA system. © 1994 John Wiley & Sons, Inc.     

Neurotransmitter release: development of a theory for total release based on kinetics

According to the calcium-voltage hypothesis for the control of neurotransmitter release, a molecule (or molecular complex) must be activated by membrane depolarization, after which the activated molecule can bind calcium and initiate release. In this study, we have examined properties of the kinetics of phasic release resulting from a set of differential equations that characterize the calcium-voltage hypothesis. It was found that, in accord with experiments, an important feature is the approximate constancy of the shape of the graph for the kinetics of phasic release at various depolarizations and extracellular calcium concentrations. The shape constancy allowed us to obtain an explicit and relatively simple analytical formula for the total transmitter release (quantal content) by approximating the differential equations of the model. This formula shows a saturating sigmoidal dependence on both intracellular and extracellular calcium concentrations. The formula thus agrees with various experiments. Moreover, it agrees with, and provides meaning to, earlier phenomenological expressions for the dependence of release on calcium concentration. In particular, the formula provides an expression for the maximal release in terms of kinetic parameters from the calcium-voltage model, and thereby allows one to supplement earlier kinetic tests of the calcium-voltage hypothesis with further tests focused upon the dependence of total release on depolarization.     

Mathematics of band centrifugation: Concentration-independent sedimentation and diffusion in shallow density gradients

Integral expressions for concentration as a function of time and distance are derived from the continuity equation for centrifugation in a sector-shaped cell for a macro-molecular solute initially contained in a finite upper layer and a solute of low molecular weight in the supporting liquid. Computer patterns based on the sedimentation and diffusion coefficients of sucrose and of spherical and randomly coiled model solutes illustrate: (1) the time course of redistribution of both banded and supporting solutes from initial uniform concentrations; (2) the influence of the initial concentration, width, and solute concentration of the upper band; and (3) the effect of restricted diffusion at the meniscus on subsequent band shape. A Gaussian, approximation to band shape is derived and graphically tested. Rapid methods, not requiring computers, are out lined for the estimation of sedimentation and diffusion coefficients, where their concentration dependence is negligible, by band centrifugtion. The theoretical resolution of mixtures attainable by this technique is compared with moving-boundary centrifugation, with the use of both integral (interferotmetric or absorption) and derivative (schlieren) optics.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, R_e. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the ‘equilibrium partition model’, the dependence of the ‘solubilizing detergent concentration’ on the lipid concentration intersects with the lipid axis at −1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical (‘solubilizing’) effective ratio depends upon the nature of detergent less than any of these two factors.     

A Quantitative Model for the Dependence of Solute Permeability on Peptide and Cholesterol Content in Biomembranes

The influence of varying concentrations of a transmembrane peptide, gramicidin A (gA), and cholesterol (Chol) on the passive permeation of p-methylhippuric acid (MHA) and α-carbamoyl-p-methylhippuric acid (CMHA) across egg-lecithin membranes (EPC) has been investigated in vesicle efflux experiments. Incorporation of 0.25 volume fraction of gA in its nonchannel conformation increased the permeability coefficient (P _m ) for CMHA by a factor of 6.0 ± 1.8 but did not alter P _m for MHA, a more lipophilic permeant. In contrast, incorporation of 0.26 volume fraction Chol with no added protein decreased the P _m values for both CMHA and MHA by similar factors of 4.2 ± 1.1 and 3.5 ± 1.2, respectively. A quantitative structure-transport model has been developed to account for the dependence of P _m on the membrane concentrations of gA and Chol in terms of induced changes in both membrane chain ordering and hydrophobicity. Chain ordering is assumed to affect P _m for both permeants similarly since they are comparable in molecular size, while changes in P _m ratios in the presence of gA or Chol are attributed to alterations in membrane hydrophobicity. Changes in lipid chain ordering were detected by monitoring membrane fluidity using fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene incorporated into the membranes. The influence of additives on membrane hydrophobicity, which governs P _m ratios through effects on solute partitioning into the barrier domain, were rationalized within the framework of regular solution theory using solubility parameters as a measure of membrane hydrophobicity. Fits of the P _m ratios using the theoretical model yielded solubility parameters for gA and Chol in EPC membranes of 13.2 and 7.7 (cal/ml)^1/2, respectively, suggesting that gA decreases the barrier domain hydrophobicity while Chol has a minimal effect on barrier hydrophobicity. After correcting for barrier domain hydrophobicity, permeability decrements due to membrane ordering induced by gA or Chol were found to exhibit a strong correlation with membrane order as predicted by free-surface-area theory, regardless of whether gA or Chol is used as the ordering agent. Received: 20 December 1999/Revised: 21 June 2000     

Mechanisms of alveolar protein clearance in the intact lung

Transport of protein across the alveolar epithelial barrier is a critical process in recovery from pulmonary edema and is also important in maintaining the alveolar milieu in the normal healthy lung. Various mechanisms have been proposed for clearing alveolar protein, including transport by the mucociliary escalator, intra-alveolar degradation, or phagocytosis by macrophages. However, the most likely processes are endocytosis across the alveolar epithelium, known as transcytosis, or paracellular diffusion through the epithelial barrier. This article focuses on protein transport studies that evaluate these two potential mechanisms in whole lung or animal preparations. When protein concentrations in the air spaces are low, e.g., albumin concentrations <0.5 g/100 ml, protein transport demonstrates saturation kinetics, temperature dependence indicating high energy requirements, and sensitivity to pharmacological agents that affect endocytosis. At higher concentrations, the protein clearance rate is proportional to protein concentration without signs of saturation, inversely related to protein size, and insensitive to endocytosis inhibition. Temperature dependence suggests a passive process. Based on these findings, alveolar albumin clearance occurs by receptor-mediated transcytosis at low protein concentrations but proceeds by passive paracellular mechanisms at higher concentrations. Because protein concentrations in pulmonary edema fluid are high, albumin concentrations of 5 g/100 ml or more, clearance of alveolar protein occurs by paracellular pathways in the setting of pulmonary edema. Transcytosis may be important in regulating the alveolar milieu under nonpathological circumstances. Alveolar degradation may become important in long-term protein clearance, clearance of insoluble proteins, or under pathological conditions such as immune reactions or acute lung injury. acute respiratory distress syndrome; endocytosis; diffusion; protein transport pulmonary edema     

Comparison of dwarf bamboos (Indocalamus sp.) leaf parameters to determine relationship between spatial density of plants and total leaf area per plant

The relationship between spatial density and size of plants is an important topic in plant ecology. The self‐thinning rule suggests a ?3/2 power between average biomass and density or a ?1/2 power between stand yield and density. However, the self‐thinning rule based on total leaf area per plant and density of plants has been neglected presumably because of the lack of a method that can accurately estimate the total leaf area per plant. We aimed to find the relationship between spatial density of plants and total leaf area per plant. We also attempted to provide a novel model for accurately describing the leaf shape of bamboos. We proposed a simplified Gielis equation with only two parameters to describe the leaf shape of bamboos one model parameter represented the overall ratio of leaf width to leaf length. Using this method, we compared some leaf parameters (leaf shape, number of leaves per plant, ratio of total leaf weight to aboveground weight per plant, and total leaf area per plant) of four bamboo species of genus Indocalamus Nakai (I. pedalis (Keng) P.C. Keng, I. pumilus Q.H. Dai and C.F. Keng, I. barbatus McClure, and I. victorialis P.C. Keng). We also explored the possible correlation between spatial density and total leaf area per plant using log‐linear regression. We found that the simplified Gielis equation fit the leaf shape of four bamboo species very well. Although all these four species belonged to the same genus, there were still significant differences in leaf shape. Significant differences also existed in leaf area per plant, ratio of leaf weight to aboveground weight per plant, and leaf length. In addition, we found that the total leaf area per plant decreased with increased spatial density. Therefore, we directly demonstrated the self‐thinning rule to improve light interception.     

The ELLIPS suite of macromolecular conformation algorithms

This paper describes a series of four programmes for the PC based on ellipsoidal representations of macromolecular shape in solution using Universal shape functions. ELLIPS1 is based on simple ellipsoid of revolution models (where two of the three axes of the ellipsoid are fixed equal to each other). If the user types in a value for a shape function from sedimentation or other types of hydrodynamic measurement, it will return a value for the axial ratio of the ellipsoid. ELLIPS2 is based on the more general triaxial ellipsoid with the removal of the restriction of two equal axes. The user enters the three semi-axial dimensions of the molecule or the equivalent two axial ratios and ELLIPS2 returns the value of all the hydrodynamic shape functions. It also works of course for ellipsoids of revolution. ELLIPS3 and ELLIPS4 do the reverse of ELLIPS2, that is they both provide a method for the unique evaluation of the triaxial dimensions or axial ratios of a macromolecule (and without having to guess a value for the so-called „hydration”) after entering at least three pieces of hydrodynamic information: ELLIPS3 requires EITHER the intrinsic viscosity with the second virial coefficient (from sedimentation equilibrium, light scattering or osmometry) and the radius of gyration (from light or x-ray scattering) OR the intrinsic viscosity with the concentration dependence term for the sedimentation coefficient and the (harmonic mean) rotational relaxation time from fluorescence depolarisation measurements. ELLIPS4 evaluates the tri-axial shape of a macromolecule from electro-optic decay based Universal shape functions using another Universal shape function as a constraint in the extraction of the decay constants. Accepted: 1 November 1996     

Effect of sphingosine and phytosphingosine ceramide ratio on lipid arrangement and barrier function in skin lipid models

The lipids in the uppermost layer of the skin, the stratum corneum (SC), play an important role in the skin barrier function. The three main subclasses in the SC lipid matrix are ceramides (CER), cholesterol, and free fatty acids. In inflammatory skin diseases, such as atopic dermatitis and psoriasis, the SC lipid composition is modulated compared to the composition in healthy SC. One of the main alterations is the molar ratio between the concentration of CER N-(tetracosanoyl)-sphingosine (CER NS) and CER N-(tetracosanoyl)-phytosphingosine (CER NP), which correlated with an impaired skin barrier function. In the present study, we investigated the impact of varying the CER NS:CER NP ratios on the lipid organization, lipid arrangement, and barrier functionality in SC lipid model systems. The results indicate that a higher CER NS:CER NP ratio as observed in diseased skin did not alter the lipid organization or lipid arrangement in the long periodicity phase encountered in SC. The trans-epidermal water loss, an indication of the barrier functionality, was significantly higher for the CER NS:CER NP 2:1 model (mimicking the ratio in inflammatory skin diseases) compared to the CER NS:CER NP 1:2 ratio (in healthy skin). These findings provide a more detailed insight into the lipid organization in both healthy and diseased skin and suggest that in vivo the molar ratio between CER NS:CER NP contributes to barrier impairment as well but might not be the main factor.     

Inferring the temperature dependence of population parameters: the effects of experimental design and inference algorithm

Understanding and quantifying the temperature dependence of population parameters, such as intrinsic growth rate and carrying capacity, is critical for predicting the ecological responses to environmental change. Many studies provide empirical estimates of such temperature dependencies, but a thorough investigation of the methods used to infer them has not been performed yet. We created artificial population time series using a stochastic logistic model parameterized with the Arrhenius equation, so that activation energy drives the temperature dependence of population parameters. We simulated different experimental designs and used different inference methods, varying the likelihood functions and other aspects of the parameter estimation methods. Finally, we applied the best performing inference methods to real data for the species Paramecium caudatum. The relative error of the estimates of activation energy varied between 5% and 30%. The fraction of habitat sampled played the most important role in determining the relative error; sampling at least 1% of the habitat kept it below 50%. We found that methods that simultaneously use all time series data (direct methods) and methods that estimate population parameters separately for each temperature (indirect methods) are complementary. Indirect methods provide a clearer insight into the shape of the functional form describing the temperature dependence of population parameters; direct methods enable a more accurate estimation of the parameters of such functional forms. Using both methods, we found that growth rate and carrying capacity of Paramecium caudatum scale with temperature according to different activation energies. Our study shows how careful choice of experimental design and inference methods can increase the accuracy of the inferred relationships between temperature and population parameters. The comparison of estimation methods provided here can increase the accuracy of model predictions, with important implications in understanding and predicting the effects of temperature on the dynamics of populations.     

Fluorescence energy transfer in one dimension: Frequency-domain fluorescence study of DNA–fluorophore complexes

The theory for the salt dependence of the free energy, entropy, and enthalpy of a polyelectrolyte in the PB (PB) model is extended to treat the nonspecific salt dependence of polyelectrolyte–ligand binding reactions. The salt dependence of the binding constant (K) is given by the difference in osmotic pressure terms between the react ants and the products. For simple 1-1 salts it is shown that this treatment is equivalent to the general preferential interaction model for the salt dependence of binding [C. Anderson and M. Record (1993) Journal of Physical Chemistry, Vol. 97, pp. 7116–7126]. The salt dependence, entropy, and enthalpy are compared for the PB model and one specific form of the preferential interaction coefficient model that uses counterion condensation/limiting law (LL) behavior. The PB and LL models are applied to three ligand–polyelectrolyte systems with the same net ligand charge: a model sphere–cylinder binding reaction, a drug–DNA binding reaction, and a protein–DNA binding reaction. For the small ligands both the PB and limiting law models give (ln K vs. In [salt]) slopes close in magnitude to the net ligand charge. However, the enthalpy/entropy breakdown of the salt dependence is quite different. In the PB model there are considerable contributions from electrostatic enthalpy and dielectric (water reorientation) entropy, compared to the predominant ion cratic (release) entropy in the limiting law model. The relative contributions of these three terms in the PB model depends on the ligand: for the protein, ion release entropy is the smallest contribution to the salt dependence of binding. The effect of three approximations made in the LL model is examined: These approximations are (1) the ligand behaves ideally, (2) the preferential interaction coefficient of the polyelectrolyte is unchanged upon ligand binding, and (3) the polyelectrolyte preferential interaction coefficient is given by the limiting law/counterion-condensation value. Analysis of the PB model shows that assumptions 2 and 3 break down at finite salt concentrations. For the small ligands the effects on the slope cancel, however, giving net slopes that are similar in the PB and LL models, but with a different entropy/enthalpy breakdown. For the protein ligand the errors from assumptions 2 and 3 in the LL model do not cancel. In addition, the ligand no longer behaves ideally due to its complex structure and charge distribution. Thus for the protein the slope is no longer related simply to the net ligand charge, and the PB model gives a much larger slope than the LL model. Additionally, in the PB model most of the salt dependence of the protein binding comes from the change in ligand activity, i.e. from nonspecific anion effects, in contrast to the small ligand case. While the absolute binding is sensitive to polyelectrolyte length, little length effect is seen on the salt dependence for the small ligands at 0.1M salt, and for lengths > 60 Å. Almost no DNA length dependenceis seen in the salt dependence of the protein binding, since this is determined primarily by the protein, not the DNA. © 1995 John Wiley & Sons, Inc.     

Asymmetry of the gramicidin channel in bilayers of asymmetric lipid composition: I. Single channel conductance

Summary Gramicidin-doped asymmetric bilayers made by the Montal-Mueller method exhibited an asymmetric current-voltage relationship. The asymmetric conductance was shown to be the product of two components, a rectifying single-channel conductance and an asymmetric voltage dependence of the reaction which leads to the conducting channel. The single-channel conductance was asymmetric in both asymmetric bilayers made of charged lipids and asymmetric bilayers made only of neutral lipids. The single-channel asymmetry decreased with increasing ion concentration. From the comparison of the singlechannel conductance in symmetric and asymmetric bilayers and the dependence of the asymmetry on the solution ion concentrations, it was concluded that (1) the rate of ion entry into the channel is dependent on the lipid composition of the membrane and is asymmetric in asymmetric bilayers; (2) the entry step is rate determining at low ion concentrations; and (3) at higher ion concentrations the rate-determining step is the translocation across the main barrier in the membrane; and this translocation appears insensitive to lipid asymmetry.     

Hydroxyethyl Starch (HES 130/0.4) Impairs Intestinal Barrier Integrity and Metabolic Function: Findings from a Mouse Model of the Isolated Perfused Small Intestine

BackgroundThe application of hydroxyethyl starch (HES) for volume resuscitation is controversially discussed and clinical studies have suggested adverse effects of HES substitution, leading to increased patient mortality. Although, the intestine is of high clinical relevance and plays a crucial role in sepsis and inflammation, information about the effects of HES on intestinal function and barrier integrity is very scarce. We therefore evaluated the effects of clinically relevant concentrations of HES on intestinal function and barrier integrity employing an isolated perfused model of the mouse small intestine.MethodsAn isolated perfused model of the mouse small intestine was established and intestines were vascularly perfused with a modified Krebs-Henseleit buffer containing 3% Albumin (N=7) or 3% HES (130/0.4; N=7). Intestinal metabolic function (galactose uptake, lactate-to-pyruvate ratio), edema formation (wet-to-dry weight ratio), morphology (histological and electron microscopical analysis), fluid shifts within the vascular, lymphatic and luminal compartments, as well as endothelial and epithelial barrier permeability (FITC-dextran translocation) were evaluated in both groups.ResultsCompared to the Albumin group, HES perfusion did not significantly change the wet-to-dry weight ratio and lactate-to-pyruvate ratio. However, perfusing the small intestine with 3% HES resulted in a significant loss of vascular fluid (p<0.01), an increased fluid accumulation in the intestinal lumen (p<0.001), an enhanced translocation of FITC-dextran from the vascular to the luminal compartment (p<0.001) and a significantly impaired intestinal galactose uptake (p<0.001). Morphologically, these findings were associated with an aggregation of intracellular vacuoles within the intestinal epithelial cells and enlarged intercellular spaces.ConclusionA vascular perfusion with 3% HES impairs the endothelial and epithelial barrier integrity as well as metabolic function of the small intestine.     

Effects of membrane reference state on shape memory of a red blood cell

By using a three-dimensional continuum model, we simulate the shape memory of a red blood cell after the remove of external forces. The purpose of this study is to illustrate the effect of membrane reference state on cell behavior during the recovery process. The reference state of an elastic element is the geometry with zero stress. Since the cell membrane is composed of cytoskeleton and lipid bilayer, both the reference states of cytoskeleton (RSC) and lipid bilayer (RSL) are considered. Results show that a non-spherical RSC can result in shape memory. The energy barrier due to non-spherical RSC is determined by the ratio of the equator length to the meridian length of the RSC. Thus different RSCs can have similar energy barrier and leading to identical recovery response. A series of simulations of more intermediate RSCs show that the recovery time scale is inversely proportional to the energy barrier. Comparing to spherical RSL, a spheroid RSL contributes to the energy barrier and recovery time. Furthermore, we observe a folding recovery due to the biconcave RSL which is different from the tank treading recovery. These results may motivate novel numerical and experimental studies to determine the exact RSC and RSL.     

Cell wall proteomics contributes to explore the functional proteins of Brachypodium distachyon grains

The plant cell wall is the first barrier in response to external stimuli and cell wall proteins (CWPs) can play an important role in the modulation of plant growth and development. In the past 10 years, the plant cell wall proteomics has increasingly become a very active research filed, which provides a broader understanding of CWPs for people. The cell wall proteome of Arabidopsis, rice, and other model plants has begun to take shape, and proteomic technology has become an effective way to identify the candidate functional CWPs in large scale. The challenging work of Francin‐Allami et al. (Proteomics 2015, 15, 2296–2306) is a vital step toward building the most extensive cell wall proteome of a monocot species. They identified 299 cell wall proteins in Brachypodium distachyon grains, and also compared the grain cell wall proteome with those of B. distachyon culms and leaves, which provides a new perspective for further explaining the plant cell wall structures and remodeling mechanism.     

Protein shape change has a major effect on the gating energy of a mechanosensitive channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k_BT to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k_BT on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.     

Structure of the squid axon membrane as derived from charge-pulse relaxation studies in the presence of absorbed lipophilic ions

Summary Charge-pulse relaxation studies were performed on squid giant axons in the presence of membrane absorbed lipophilic anions, dipicrylamine (DPA) and tetraphenylborate (TPhB), and of specific blockers of sodium and potassium active currents. With the instrumentation used in this work a time resolution of 5 to 10 sec was easily obtained without any averaging, although the voltage relaxations were always smaller than 5 mV in amplitude in order to keep the membrane voltage in a range where the used theory cyn be linearized. Two well distinguishable linear relaxations were invariably observed in the presence of the lipophilic anions. With DPA the fast relaxation (time constants between 8 and 70 sec) was attributed to the redistribution of the lipophilic ions within the membrane following the change in membrane potential. The long relaxation process (time constant in the millisecond range) corresponds to the normal voltage relaxation of the passive squid axon membrane slightly modified by the process of redistribution of the extrinsic ions.The results support the same model for the translocation of lipophilic ions within the nerve membrane proposed earlier for artificial lipid bilayers. The fit of the data with a single barrier model yields the translocation rate constant,K, and the total concentration,N _t , of membrane absorbed ions, from which the membrane-solution partition coefficient, , can be derived. Both for DPA and TPhB,K had values close to those measured for solvent-free artificial lipid bilayers. The axon membrane appears as fluid mosaic membrane with a thickness of about 2.5 nm for the lipid bilayer part.In axons treated with DPA the dependence of relaxation data upon the holding membrane potential, , provided information on the asymmetry of the membrane structure. The data were best fitted by assuming that nearly 100% of the membrane potential drops between the two free energy minima where the extrinsic ions are located, indicating that these minima lie very close to the membrane-solution interfaces, in the region of the phospholipid polar heads. The asymmetry voltage,E _o, at which the extrinsic ions are expected to be equally distributed between the two sides of the membrane was found to range between –35 and –65 mV (inside negative), depending on the assumed shape of the free energy barrier describing the ion translocation process. This voltage is of the same sign and of the same order of magnitude as the equilibrium voltages for the open-close transitions of the gates of sodium and potassium channels, suggesting that all these voltages result from the same membrane asymmetry. A similar analogy was found between the asymmetry of the free energy barrier which best fitted DPA relaxation data and the asymmetrical voltage dependence of the gating of ionic channels. Our data were best fitted by assuming that about 70% of the potential drop occurs between the free energy minimum on the intracellular membrane face and the top of the barrier.     

Calcium binding to bone gamma-carboxyglutamic acid protein from calf studied by 43Ca NMR

Calcium binding to bone gamma-carboxyglutamic acid protein (BGB) from calf has been studied using 43Ca NMR. The temperature dependence of the 43Ca NMR signal has been used to calculate the calcium ion exchange rate, koff. The dependence of the 43Ca NMR band shape on the [Ca2+]/[BGP] ratio fits well to a chemical equilibrium model having a single Ca2+-binding site with an association constant in the range of 5 X 10(3)-1 X 10(5) M-1. The pH dependence of the 43Ca NMR line-width shows a single apparent pKa value of 5.1.