Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT(1A) receptor

Deuterium oxide solutions of a triple-helical polysaccharide schizophyllan, undergoing an order-disorder transition centered around 17 degrees C, were studied by the time-domain reflectometry (TDR) to obtain dielectric dispersions in the solution and frozen states. In the solution state, the dispersion below the transition temperature is resolved in three dispersions (relaxation times at 0 degrees C) ascribed to side chain glucose residue (1; 102 ns), structured water (s; 2.0 ns) and bulk water (h), respectively, from low to high frequencies. Bulk water is divided into slow water (h2; 0.04 ns) and free or pure water (h1; 0.02 ns). Above the transition temperature structured water almost disappears and is compensated by slow water. Structured water is similar to bound water for proteins but different from it because of this transition behavior. Another dispersion (l) seen at the lowest frequency is assigned to the rotation of side-chain glucose residue coupled with hydrated water. Parts of this dispersion and structured water are suggested to constitute bound water. In the frozen state were observed a major dispersion (h; 0.14 ns) and a minor one (m; 28 ns), which were ascribed to considerably mobile and less mobile waters. They are similar to but not exactly the same as that for unfreezable water in bovine serum albumin solutions argued by Miura et al. (Biopolymers, 1995, Vol. 36, p. 9). Water is molded into different structures by the triple helix.     

Static water structure detected by heat capacity measurements on aqueous solutions of a triple-helical polysaccharide schizophyllan

Heat capacity measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan by precision adiabatic calorimetry over a wide range of concentrations 30.45-90.93 wt % at temperatures between 5 and 315 K. The heat capacity curves obtained were divided into four groups depending on the weight fraction of schizophyllan w regions I-IV. In region I, triple-helices with the sheath of bound water, structured water, and loosely structured water forming layers around the helix core are embedded in free water. In region II, there is no free water, and loosely structured water decreases until it vanishes, but structured water stays constant with increasing w. In region III, bound water remains unaffected, but structured water decreases with increasing w by overlapping each other. Finally, in region IV, only schizophyllan and bound water exist, the latter decreasing upon increasing w. The maximum thickness of each layer is 0.18(3) nm for bound water, 0.13(4) nm for structured water, and 0.23(6) nm for loosely structured water, and these layers of water are at the enthalpy levels of 53%, 93.7%, and nearly 100%, respectively, between ice (0%) and free water (100%).     

Ordering in aqueous polysaccharide solutions. II. Optical rotation and heat capacity of aqueous solutions of a triple-helical polysaccharide schizophyllan

Deuterium oxide solutions of schizophyllan, a triple-helical polysaccharide, undergoing an order-disorder transition centered at 17 degrees C, were studied by optical rotation (OR) and heat capacity (C(p)) to elucidate the molecular mechanism of the transition and water structure in the solution and frozen states. The ordered structure at low temperature consisted of the side chains and water in the vicinity forming an ordered hydrogen-bonded network surrounding the helix core and was disordered at higher temperature. In the solution state appeared clearly defined transition curves in both the OR and C(p) data. The results for three samples of different molecular weights were analyzed theoretically, treating this transition as a typical linear cooperative transition from the ordered to disordered states and explained quantitatively if the molecular weight polydispersity of the sample was considered. The excess heat capacity C(EX)(p) defined as the C(p) minus the contributions from schizophyllan and D(2)O was estimated. In the frozen state it increased with raising temperature above 150 K until the mixture melted. This was compared with the dielectric increment observed in this temperature range and ascribed to unfreezable water. From the heat capacity and dielectric data, unfreezable water is mobile but more ordered than free water. In the solution state, the excess heat capacity originates from the interactions of D(2)O molecules as bound water and structured water, and so forth. Thus the schizophyllan triple helix molds water into various structures of differing orders in solution and in the solid state.     

The Dielectric Relaxation of Water in Plant Tissue

Thermally stimulated depolarization current (TSDC) measurementson plant leaves and stems of six different species in the temperaturerange of 77–300 K revealed the existence of three differentdispersions. The first dispersion at low temperatures, whichis attributed to the relaxation of loosely bound water moleculeswas studied in detail in an attempt to obtain information onthe possible structures of water in plant tissue. Its characteristicsdiffer for various plant tissues, indicating a different organizationof water in those plant tissues. The dispersion can be describedby a continuous distribution of relaxation times t with boththe activation energy W and the pre-exponential factor To inthe Arrhenius equation being distributed parameters. The spectrumof W and T_o was determined for E. globulus and O. europaea leafsamples. The mean values of T and W are larger and that of T_osmaller than the corresponding values for free (bulk) water.The results favour a model of the organization of water in clustersrather than in multilayers and indicate a stronger binding ofwater in living systems. Key words: Dielectric relaxation, distribution of relaxation times, free and bound water     

A Dielectric Study of the State of Water in Plant Stems

Pissis, P., Anagnostopoulou-Konsta, A. and Apekis, L. 1987.A dielectric study of the state of water in plant stems.—J.exp. Bot. 38: 1528–1540. We report on thermally stimulated depolarization current (TSDC)measurements on plant stems of six different species in thetemperature range of 77–300 K and over a wide range ofwater contents, in an attempt to determine the binding modesof water molecules. The measurements revealed the existenceof three dielectric dispersions. The first dispersion at lowtemperatures is attributed to the reorientation of loosely boundwater molecules. The origin of the second dispersion at intermediatetemperatures is not yet clear. It is either due to the reorientationof tightly bound water molecules or to the relaxation of somebotanical materials rather than water. Finally, the third dispersion,at high temperatures, is attributed to water-assisted spacecharge polarization connected with dc ionic conductivity. Theamount of tightly bound water is estimated to be about 0·2-g-g¹of dry material or about 30% of the total water content, therest being mainly loosely bound water. Little or none of thewater behaves dielectrically like pure water. Key words: Free and bound water, dielectric properties, water content.     

Electron spin relaxation time of Fe(III) in high spin heme proteins.

The electron spin relaxation time of high spin Fe(III), taus, was determined from the frequency dependence (5-100 MHz) of the longitudinal proton relaxation rates of water in solutions of catalase, metmyoglobin and acid ferricytochrome c. In all three high-spin heme proteins the relaxation rates incrased below 25 MHz, while no frequency dependence was observed above that frequency. The results are interpreted by assuming that taus, which modulates the dipolar interaction between the unpaired electrons of the iron and the water protons, is frequently independent. Its value was determined to be (6 +/- 1) - 10(-11) s.     

Initial stages of lichen hydration observed by proton magnetic relaxation

The hydration of selected lichens ( Cladonia mitis , Cladonia bellidiflora , Cetraria islandica , Parmelia saxatilis , and Xanthoria parietina ) was investigated using gravimetry and proton magnetic free induction decays (FIDs).
The hydration from gaseous phase and dehydration to gaseous phase showed first-order kinetics. The amount of water which was non-removable in the air-dry state (relative humidity p / p ₀=9%) did not depend significantly on the lichen species and was found to be 5·6±1·0% of the d. wt.
The proton FID Gaussian component from the solid matrix of thallus structure, and two (or, depending on lichen species, one averaged) liquid signals coming from water tightly bound on the surface of thallus solid matrix and from loosely bound or free water, were recorded. The bound-water component was distinguished by its motional properties and by its proximity to endogenous paramagnetic centres present in solid matrix (presumably PS II reaction centres of the photobiont). Mild dehydration (from gaseous phase) could completely remove the loosely bound water fraction, leaving the system below the water percolation threshold and below the water clustering point, emphasizing the passivity of lichen response to desiccation shock. In the species in which the one average liquid component was recorded, bound water behaved similarly.
The hydration at which free water pool vanishes (Δ M / m ₀) and the relative (scaled to water) proton densities of solid matrix of lichen (β) were evaluated for all lichens investigated.     

Index to Authors,Volume 7

Abstract

The thermally stimulated depolarization current (TSDC) measurements in frozen aqueous solutions, gels and solid layers of NaDNA show typically up to three dipolar overlapping peaks in the low-temperature range of 80—;150 K. Up to four discrete relaxation peaks have been observed at higher temperatures above 150 K. The low-temperature TSDC peaks are due to the dipolar relaxations of free and loosely bound water which crystallizes. Part of bound water especially in the first hydration shell of DNA molecule is at low temperatures in the form of glass. The transition of this glass from solidlike behavior to liquidlike behavior observed mainly in gels and solid samples is associated with a previously founded TSDC relaxation peak. The peak is at its maximum at 165- 250 K depending on the sample humidity. Existence of this relaxation in the samples with water contents in a broad range confirms, that the slowly relaxing shell (minimally 5–7 water molecules/nucleotide) closely associated with DNA double helix retains its characteristics. Also another peak of the high-temperature band at 180–205 K which was observed in the samples at hydration 2–1800 g H₂O/g dry NaDNA is due to a relaxation in the sample volume. At the highest temperatures relax the space charges trapped at the electrodes.     

Frequency dependence of water proton longitudinal NMR relaxation times in mouse tissues around the freezing transition

Water proton longitudinal NMR relaxation times were measured in various tissues of healthy and tumor-bearing mice. Measurements were performed as a function of the Larmor frequency nu in the range 6-90 MHz, and at two temperatures (theta + and theta -) bracketing the 'freezing transition', at which the major part of the water signal disappears. At both temperatures, 1/T1 behaves according to: 1/T1 = A/square root nu + B A and B are obtained at theta + and theta -, and yield the proportion of bound water, which is convincingly identified with non-freezable water. The proportions found lie around 6% for tumors and 12% for other tissues. Discrimination between tissues via T1 is demonstrated to be essentially due to the bound water proportion. Bound water on the one hand and free water on the other hand behave similarly in all tissues including tumors. The activation energy for free water is found to be identical to that of pure water, although relaxation times are markedly different. It is noticed that determining the bound water proportion by signal intensity measurements at theta + and theta - is less reliable than by the T1 method.     

Water structure in modified bovine plasma albumin (BPA) gel and bovine mercaptalbumin solution. 1H-n.m.r. studies

Bovine plasma albumin Fr. V (BPA) has been known to contain small amounts of proteolytic enzyme. Wilson & Foster (1971) found a very limited and specific cleavage of BPA catalyzed by the enzyme with BPA in the F-form near pH 3.8, resulting in the formation of partially hydrolyzed BPA (BPA*). BPA* had a tendency to form a transparent gel at pD 4.0 (pD range of the F-form) above 8%, though proteolytic enzyme-free bovine mercaptalbumin (BMA) was in a transparent solution at pD 4.0 even at 12%. Water structures of the F-form of BMA in the solution state and of BPA* in the gel state were studied by measuring 1H-n.m.r. spectra, spin-lattice relaxation time (T1) and cross relaxation time (TIS) between irradiated and observed protons. Protein concentration-dependent changes of T1 of water protons indicated that the amount of hydrated water of BPA* in the gel state is far greater than that of the F-form of BMA in the solution state. TIS values from protein protons to water protons also indicated a large amount of hydration of BPA*, strong interaction between water and BPA* and rapid exchange between bound and bulk water in the gel state.     

Nuclear magnetic relaxation by the manganese in aqueous suspensions of chloroplasts

Proton and oxygen-17 NMR relaxation rate (T1-1 and T2-1) data are presented for aqueous suspensions of dark-adapted chloroplasts. It is concluded from the dependence of the proton relaxation rates (PRR) upon Mn concentration that T1-1 and T2-1 are determined largely by the loosely bound Mn present in the chloroplast membranes. The frequency and temperature dependences of PRR are characteristic of Mn(II). The effects of oxidants (e.g., ferricyanide) and reductants (e.g., tetraphenylboron) on the PRR indicate that only about one-third to one-fourth of the loosely bound Mn is present in the dark-adapted chloroplasts as Mn(II), the remainder being in a higher oxidation state(s), probably Mn(III). The frequency dependence of the PRR for the chloroplast suspensions was fitted by a simplified form of the Solomon-Bloembergen-Morgan equations, and the following parameters were obtained: tauS = (1.1 +/- 0.1) X 10(-8) S; tauM = (2.2 +/- 0.2) X 10(-8) S; and B = (0.9 +/- 0.09) X 10(19). The oxygen-17 T1 and T2 data for suspensions before and after treatment with a detergent are consistent with the location of the manganese in the interior of the thylakoids. An analysis of the relaxation rates shows that the average lifetime of a water molecule inside a thylakoid is greater than 1 ms.     

The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.     

The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.     

Fatty acid binding sites of serum albumin probed by non-linear spin-label EPR

A novel form of non-linear EPR spectroscopy, viz. the first harmonic absorption spectrum recorded in phase quadrature with respect to the Zeeman field modulation, is used here to investigate spin-lattice relaxation enhancements of nitroxide spin labels bound to serum albumin that are induced by spin-spin interactions with aqueous paramagnetic ions. The advantage of this EPR method is that it is directly sensitive to spin-lattice relaxation and affected relatively little by other spectral parameters (Livshits et al., J. Magn. Reson. 133 (1998) 79-91). Relaxation enhancements by ferricyanide of bound fatty acids (n-SASL) spin-labelled at different positions, n, in the chain are compared with those of different maleimide spin label derivatives attached at the single free -SH group, as well as with those of the spin labels free in solution. It was found that: (1) the encounter frequency of ferricyanide with 5-SASL and 12-SASL bound to serum albumin is more than two times less than that with 16-SASL; (2) the accessibility of ferricyanide to 16-SASL is comparable to that of the more immobilised covalently bound spin labels; and (3) the absolute values of the encounter frequencies for the bound spin-labelled fatty acids are approximately a factor of ten smaller than for the corresponding free spin labels, but the latter show a dependence on position of labelling that is similar to the bound labels. A kinetic scheme that is consistent with these relative differences involves rapid reversible transitions between an 'open' and 'closed' state, in which interaction with aqueous paramagnetic agents is possible only in the 'open' state. The equilibrium strongly favours the 'closed' state, which is further enhanced at low temperatures.     

Multinuclear NMR study of enzyme hydration in an organic solvent

Multinuclear NMR spectroscopy has been used to study water bound to subtilisin Carlsberg suspended in tetrahydrofuran (THF), with the water itself employed as a probe of the hydration layer's physicochemical and dynamic characteristics. The presence of the enzyme did not affect the intensity, chemical shift or linewidth of water (up to 8% v/v) added to THF, as measured by 17O- and 2H-NMR. This finding suggests that hydration of subtilisin can be described by a three-state model that includes tightly bound, loosely bound, and free water. Solid-state 2H-NMR spectra of enzyme-bound D2O support the existence of a non-exchanging population of tightly bound water. An important implication is that the loosely-bound water is the same as free water from an NMR viewpoint. This loosely bound water must also be the water responsible for the large increase in catalytic activity observed in previous hydration studies.     

An evaluation of the hydration of lysozyme by an NMR titration method

In this study a new titration method is proposed to study the motional properties of water molecules in conjunction with globular proteins using proton NMR relaxation measurements. The method was applied to the study of the interaction of water with lysozyme and allowed identification of four water fractions-superbound water, polar-bound water, structured water and bulk water - in exchanged equilibrium. The titration demonstrated that 193 water molecules are hydrogen bonded directly to the lysozyme molecule. The combination of structured and bound water extends to 1.4 g H2O per g lysozyme and approx. two to three layers from the surface of the macromolecule. It is proposed that this structured water is related to non-isotropic water rotation in conjunction with hydrophobic patches and directly related to 'hydrophobic bonding' changes. Water amounts greater than 1.4 g H2O per g lysozyme are sufficiently distant from the macromolecule for motion to revert to that typical of water in bulk. The typical correlation times for water motion in the four fraction are: over 10(-6) s (superbound); 10(-9) s (polar bound); 10(-11) s (structured) and 10(-12) s (bulk). These results correlate well with results from other measurement techniques found in the literature.     

Oxygen-17 and deuterium nuclear magnetic relaxation studies of lysozyme hydration in solution: field dispersion, concentration, pH/pD, and protein activity dependences

A comparison of 17O and 2H NMR relaxation rates of water in lysozyme solutions as a function of concentration, pH/pD, and magnetic field suggests that only 17O monitors directly the hydration of lysozyme in solution. NMR measurements are for the first time extended to 11.75 T. Lysozyme hydration data are analyzed in terms of an anisotropic, dual-motion model with fast exchange of water between the "bound" and "free" states. The analysis yields 180 mol "bound" water/mol lysozyme and two correlation times of 7.4 ns ("slow") and 29 ps ("fast") for the bound water population at 27 degrees C and pH 5.1, in the absence of salt, assuming anisotropic motions of water with an order parameter value for bound water of 0.12. Under these conditions, the value of the slow correlation time of bound water (7.4 ns) is consistent with the value of 8 ns obtained by frequency-domain fluorescence techniques for the correlation time associated with the lysozyme tumbling motion in solutions without salt. In the presence of 0.1 M NaCl the hydration number increases to 290 mol/mol lysozyme at pD 4.5 and 21 degrees C. The associated correlation times at 21 degrees C in the presence of 0.1 M NaCl are 4.7 ns and 15.5 ps, respectively. The value of the slow correlation time of 4.7 ns is consistent with the calculated value (4.9 ns) for the lysozyme monomer tumbling in solution. The systematic deviations of the relaxation rates, estimated with the single-exponential approximation, from the theoretical, multiexponential nuclear (I' + 1/2) spin relaxation are evaluated at various frequencies for 17O (I = 5/2) with the first-order, linear approximation (25). All NMR relaxation data for hydrated lysozymes are affected by protein activity and are sensitive both to the ionization of protein side chains and to the state of protein aggregation.     

Mobility of water bound to biological membranes. A proton NMR relaxation study.

Water proton nuclear magnetic resonance relaxation measurements have been obtained for aqueous suspensions of red cell membranes. These data support a model in which water molecules are exchanging rapidly between a bound phase with restricted motions and a free phase with dynamic properties similar to liquid water. From this model and these data, estimates are obtained for the relaxation time for bound phase water. Possible relaxation mechanisms for bound phase water are discussed and some support is found for an intermolecular interaction modulated by translational motions characterized by a diffusion constant of 10(-9) cm2/s.     

Simulated Cytoskeletal Collapse via Tau Degradation

We present a coarse-grained two dimensional mechanical model for the microtubule-tau bundles in neuronal axons in which we remove taus, as can happen in various neurodegenerative conditions such as Alzheimers disease, tauopathies, and chronic traumatic encephalopathy. Our simplified model includes (i) taus modeled as entropic springs between microtubules, (ii) removal of taus from the bundles due to phosphorylation, and (iii) a possible depletion force between microtubules due to these dissociated phosphorylated taus. We equilibrate upon tau removal using steepest descent relaxation. In the absence of the depletion force, the transverse rigidity to radial compression of the bundles falls to zero at about 60% tau occupancy, in agreement with standard percolation theory results. However, with the attractive depletion force, spring removal leads to a first order collapse of the bundles over a wide range of tau occupancies for physiologically realizable conditions. While our simplest calculations assume a constant concentration of microtubule intercalants to mediate the depletion force, including a dependence that is linear in the detached taus yields the same collapse. Applying percolation theory to removal of taus at microtubule tips, which are likely to be the protective sites against dynamic instability, we argue that the microtubule instability can only obtain at low tau occupancy, from 0.06–0.30 depending upon the tau coordination at the microtubule tips. Hence, the collapse we discover is likely to be more robust over a wide range of tau occupancies than the dynamic instability. We suggest in vitro tests of our predicted collapse.     

The reaction mechanism of paraoxon hydrolysis by phosphotriesterase from combined QM/MM simulations

Molecular dynamics simulations employing combined quantum mechanical and molecular mechanical (QM/MM) potentials have been carried out to investigate the reaction mechanism of the hydrolysis of paraoxon by phosphotriesterase (PTE). We used a dual-level QM/MM approach that synthesizes accurate results from high-level electronic structure calculations with computational efficiency of semiempirical QM/MM potentials for free energy simulations. In particular, the intrinsic (gas-phase) energies of the active site in the QM region are determined by using density functional theory (B3LYP) and second-order M?ller-Plesset perturbation theory (MP2) and the molecular dynamics free energy simulations are performed by using the mixed AM1:CHARMM potential. The simulation results suggest a revised mechanism for the phosphotriester hydrolysis mechanism by PTE. The reaction free energy profile is mirrored by structural motions of the binuclear metal center in the active site. The two zinc ions occupy a compact conformation with an average zinc-zinc distance of 3.5 +/- 0.1 A in the Michaelis complex, whereas it is elongated to 5.3 +/- 0.3 A at the transition state and product state. The substrate is loosely bound to the more exposed zinc ion (Znbeta2+) at an average distance of 3.8 A +/- 0.3 A. The P=O bond of the substrate paraoxon is activated by adopting a tight coordination to the Znbeta2+, releasing the coordinate to the bridging hydroxide ion and increasing its nucleophilicity. It was also found that a water molecule enters into the binding pocket of the loosely bound binuclear center, originally occupied by the nucleophilic hydroxide ion. We suggest that the proton of this water molecule is taken up by His254 at low pH or released to the solvent at high pH, resulting in a hydroxide ion that pulls the Znbeta2+ ion closer to form the compact configuration and restores the resting state of the enzyme.