The effects of monovalent cations Li+, Na+, K+, NH4+, Rb+ and Cs+ on the solid and solution structures of the nucleic acid components. Metal ion binding and sugar conformation.

The interactions of the monovalent ions Li+, Na+, K+, NH4+, Rb+ and Cs+ with adenosine-5'-monophosphoric acid (H2-AMP), guanosine-5'-monophosphoric acid (H2-GMP) and deoxyguanosine-5'-monophosphoric acid (H2-dGMP) were investigated in aqueous solution at physiological pH. The crystalline salts M2-nucleotide.nH2O, where M = Li+, Na+, K+ NH4+, Rb+ and Cs+, nucleotide = AMP, GMP and dGMP anions and n = 2-4 were isolated and characterized by Fourier Transform infrared (FTIR) and 1H-NMR spectroscopy. Spectroscopic evidence showed that these ions are in the form of M(H2O)n+ with no direct metal-nucleotide interaction, in aqueous solution. In the solid state, Li+ ions bind to the base N-7 site and the phosphate group (inner-sphere), while the NH4+ cations are in the vicinity of the N-7 position and the phosphate group, through hydrogen bonding systems. The Na-nucleotides and K-nucleotides are structurally similar. The Na+ ions bind to the phosphate group of the AMP through metal hydration shell (outer-sphere), whereas in the Na2-GMP, the hydrated metal ions bind to the base N-7 or the ribose hydroxyl groups (inner-sphere). The Na2-dGMP contains hydrated metal-carbonyl and metal-phosphate bindings (inner-sphere). The Rb+ and Cs+ ions are directly bonded to the phosphate groups and indirectly to the base moieties (via H2O). The ribose moiety shows C2'-endo/anti conformation for the free AMP acid and its alkali metal ion salts. In the free GMP acid, the ribose ring exhibits C3'-endo/anti conformer, while a C2'-endo/anti sugar pucker was found in the Na2-GMP and K2-GMP salts and a C3'-endo/anti conformation for the Li+, NH4+, Rb+ and Cs+ salts. The deoxyribose has C3'-endo/anti conformation in the free dGMP acid and O4'-endo/anti in the Na2-dGMP, K2-dGMP and a C3'-endo/anti for the Li+, NH4+, Rb+ and Cs+ salts. An equilibrium mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers was found for these metal-nucleotide salts in aqueous solution.     

Infrared studies of fully hydrated saturated phosphatidylserine bilayers. Effect of Li+ and Ca2+

The thermotropic phase behavior of fully hydrated Na+ and/or NH4+ salts of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) was determined by temperature-dependent infrared spectra. The molecular level properties and thermal phase behavior of DMPS-Li+ complexes were also characterized by infrared spectroscopy. With increasing concentrations of Li+, the infrared spectra reveal the appearance of a second, more ordered, lipid phase which shows a gel to liquid-crystal transition at significantly higher temperatures (75-95 degrees C) than the Na+ or NH4+ salts of DMPS (39 degrees C). Li+ binds to the phosphate and carboxylate groups of DMPS, resulting in the following changes: (1) water of hydration is lost from both the carboxylate and phosphate groups; (2) there are changes in the conformation of the glycerol backbone but not in the P-O ester bonds of the phosphate group which remain in the gauche-gauche conformation; and (3) the packing of the fatty acyl chains becomes more ordered. In addition, the properties of the DMPS-Ca2+ complex were studied by infrared spectroscopy. While the DMPS-Ca2+ complex is also characterized by rigidly packed, well-ordered fatty acyl chains, the mode of Ca2+ binding to the DMPS head groups differs significantly from that of Li+ binding. By comparison, with dry DMPS-Ca2+ [Casal, H. L., Mantsch, H. H., Paltauf, F., & Hauser, H. (1987) Biochim. Biophys. Acta (in press)], the phosphate group undergoes a conformational change, probably to the antiplanar-antiplanar conformation, and loses its water of hydration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition to a beta-sheet-rich structure in spidroin in vitro: the effects of pH and cations

Unlike man-made fibers, the silks of spiders are spun from aqueous solutions and at atmospheric pressure in a process still poorly understood. The molecular mechanism of this process involves the conversion of a highly concentrated, predominantly disordered silk protein (spidroin) into beta-sheet-rich structures. To help store and transport the spidroins in solution, as well as probably control their conversion, a liquid crystalline arrangement is established in the storage region in the ampulla and persists into the duct. Although it has been suggested that changes in the concentration of hydrogen and metal ions play a role in the formation of the solid thread, there is no reported evidence that these ions influence the secondary structure of native spidroin in solution. Here, we demonstrate that pH values between approximately 3.5 and 4.5 induce a slow change of conformation from the disordered to the beta-sheet-rich form. We also report that Al(3+), K(+), and Na(+) ions induce similar changes in structure, while Ca(2+) and Mg(2+) stabilize the predominantly disorder state of the protein. Cs(+) and Li(+) have no apparent effect. Direct volumetric and spectrophotometric titration showed a pI of 4.22 +/- 0.33 and apparent pK values of 6.74 +/- 0.71 and 9.21 +/- 0.27, suggesting a mechanism for the effect of low pH on the protein and a rationale for the observed reduction in pH in the duct. We discuss the importance of these findings for the spinning process and the active role played by the spider to alter the kinetics of the transition.     

Inhibition kinetics of hydrogen peroxide on beta-n-acetyl-D-glucosaminidase from prawn (Penaeus vannamei)

The effects of hydrogen peroxide (H2O2) on prawn NAGase activity for the hydrolysis of pNP-beta-D-GlcNAc have been studied. The results show that H2O2 can reversible inhibit the enzyme (IC50 = 0.85 M) and the inhibition is of a mixed type. The kinetics show that k+o is much larger than k+0, indicating the free enzyme is more susceptible than the enzyme-substrate complex in the H2O2 solution. It is suggested that the presence of the substrate offers marked protection against inhibition by H202. Changes of activity and conformation of the enzyme in different concentrations of H202 have been compared by measuring the fluorescence spectra and residual activity and show that the change of conformation is more rapidly than that of the residual activity, which implies that the whole conformation of the enzyme changes more rapidly than the conformation of the active centre of the enzyme in the H2O2 solution.     

Intravesicular pH changes in submitochondrial particles induced by monovalent cations: relationship to the Na+/H+ and K+/H+ antiporters

The fluorescence of internalized fluorescein isothiocyanate dextran has been used to monitor the intravesicular pH of submitochondrial particles (SMP). Respiring SMP maintain a steady-state delta pH (interior acid) that results from the inwardly directed H+ flux of respiration and an opposing passive H+ leak. Addition of K+, Na+, or Li+ to SMP results in a shift to a more alkaline interior pH (pHi) in both respiring and nonrespiring SMP. The K+-dependent change in pHi, like the K+/H+ antiport in intact mitochondria, is inhibited by quinine and by dicyclohexylcarbodiimide. The Na+-dependent reaction is only partially inhibited by these reagents. Both the Na+- and the K+-dependent pH changes are sensitive to amiloride derivatives. The Km for both Na+ and K+ is near 20 mM whereas that for Li+ is closer to 10 mM. The K+/H+ exchange reaction is only slightly inhibited by added Mg2+, but abolished when A23187 is added with Mg2+. The passive exchange is optimal at pHi 6.5 with either Na+ or K+, and cannot be detected above pHi of 7.2. Both the Na+/H+ and the K+/H+ exchange reactions are optimal at an external pH of 7.8 in respiring SMP (pHi 7.1). Valinomycin stimulates the K+-dependent pH change in nonrespiring SMP, as does nigericin. It is concluded that SMP show K+/H+ antiport activity with properties distinct from those of Na+/H+ antiport. However, the properties of the K+/H+ exchange do not correspond in all respects to those of the antiport in intact mitochondria. Donnan equilibria and parallel uniport pathways for H+ and cations appear to contribute to cation-dependent pH changes in SMP.     

An insight into the mechanism of protein separation by colloidal gas aphrons (CGA) generated from ionic surfactants

Colloidal gas aphrons (CGA), which are surfactant stabilised microbubbles, have been previously applied for the recovery of proteins from model mixtures and a few studies have demonstrated the potential of these dispersions for the selective recovery of proteins from complex mixtures. However there is a lack of understanding of the mechanism of separation and forces governing the selectivity of the separation. In this paper a mechanistic study is carried out to determine the main factors and forces influencing the selectivity of separation of whey proteins with CGA generated from ionic surfactants. Two different separation strategies were followed: (i) separation of lactoferrin and lactoperoxidase by anionic CGA generated from a solution of sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT); (ii) separation of beta-lactoglobulin by cationic CGA generated from a solution of cetyltrimethylammonium bromide (CTAB). Separation results indicate that electrostatic interactions are the main forces determining the selectivity however these could not completely explain the selectivities obtained following both strategies. Protein-surfactant interactions were studied by measuring the zeta potential changes on individual proteins upon addition of surfactant and at varying pH. Interestingly strongest electrostatic interactions were measured at those pH and surfactant to protein mass ratios which were optimum for protein separation. Effect of surfactant on protein conformation was determined by measuring the change in fluorescence intensity upon addition of surfactant at varying pH. Differences in the fluorescence patterns were detected among proteins which were correlated to differences in their conformational features which could in turn explain their different separation behaviour. The effect of conformation on selectivity was further proven by experiments in which conformational changes were induced by pre-treatment of whey (heating) and by storage at 4 degrees C. Overall it can be concluded that separation of proteins by ionic CGA is driven mainly by electrostatic interactions however conformational features will finally determine the selectivity of the separation with competitive adsorption having also an effect.     

The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies.

The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H3O+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH 4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pH-dependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A PKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H3O+ and Ca2+ result in blue-shifts in the wave-lengths of fragment-1 emission. These results are interpreted in terms of H+ - and Ca2+ - induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.     

pH-dependent conformational states of horse liver alcohol dehydrogenase.

The quenching of liver alcohol dehydrogenase protein fluorescence at alkaline pH indicates two conformational states of the enzyme with a pKa of 9.8+/-0.2, shifted to 10.6+/-0.2 in D2O. NAD+ and 2-p-toluidinonaphthalene-6-sulfonate, a fluorescent probe competitive with coenzyme, bind to the acid conformation of the enzyme. The pKa of the protein-fluorescence quenching curve is shifted toward 7.6 in the presence of NAD+, and the ternary complex formation with NAD+ and trifluoroethanol results in a pH-independent maximal quench. At pH (pD) 10.5, the rate constant for NAD+ binding was 2.6 times faster in D2O2 than in H2O due to the shift of the pKa. Based on these results, a scheme has been proposed in which the state of protonation of an enzyme functional group with a pKa of 9.8 controls the conformational state of the enzyme. NAD+ binds to the acid conformation and subsequently causes another conformational change resulting in the perturbation of the pKa to 7.6. Alcohol then binds to the unprotonated form of the functional group with a pKa of 7.6 in the binary enzyme-NAD+ complex and converts the enzyme to the alkaline conformation. Thus, at neutral pH liver alcohol dehydrogenase undergoes two conformational changes en route to the ternary complex in which hydride transfer occurs.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

Metal site conformational states of vanadyl(IV) human serotransferrin complexes.

This study was undertaken to investigate the conformational states of the two metal sites in the human serum transferrin molecule. The 9.2 GHz electron paramagnetic resonance spectra of frozen solutions of divanadyl(IV) transferrin consist of a superposition of two sets of resonances, A and B, due to the magnetically nonequivalent binding environments of the VO2+ ion. Examination of the intensities of the A and B resonances as a function of pH from 6.0 to 10.7 reveals that they arise from two conformational states of the metal sites in which the geometrical arrangement and/or identity of one or more ligands in the first coordination sphere are different. From pH 7.5 to 9.0, the metal sites exist in A and B conformations but above pH 9.0 the A conformation. This transformation is coupled to the ionization of an apparently noncoordinating protein functional group with a pK - 10.0 +/- 0.1. Below pH 7.0, binding in the B conformation is rapidly lost, driven in part by the protonation of a functional group, possibly the anion, with a pK - 6.6 +/- 0.1. In 90% D2O, this pK is elevated to 7.8 +/- 0.1. At pH 6.0 in H2O, essentially one VO2+ ion remains bound to the protein with the metal site in the A conformation. Experiments with mixed VO2+ -Fe3+ transferrin complexes indicate that the same may be true of Fe3+. At pH 10.7, a new set of VO2+ resonances, labeled C, are observed; they possibly arise from a third conformation of the metal site. One bicarbonate or corbonate is required per VO2+ ion bound to the protein. 2.7 H+ are released per VO2+ bound in either the A or B conformations. The above results are discussed in terms of the "equivalence" and "nonequivalence" of the metal sites.     

The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient

Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, Vrev, was close to the Nernst potential, EH, varying 52 mV/U pH over four delta pH units (delta pH = pHo - pHi). This result indicates that H+ channels are extremely selective, PH/PTMA > 10(7), and that both internal and external pH, pHi, and pHo, were well controlled. The H+ current amplitude was practically constant at any fixed delta pH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current- voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, gH, as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by approximately 40 mV/U pH by changes in pHi or pHo, with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as approximately 20-40(pHo-pHi) mV. If internal and external protons regulate the voltage dependence of gH gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, delta pH. We propose a simple general model to account for the observed delta pH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a conformational change resulting in the appearance of a protonation site, possibly the same one, which is accessible via the internal solution. Protonation of the internal site stabilizes the open conformation of the channel. In summary, within the physiological range of pH, the voltage dependence of H+ channel gating depends on delta pH and not on the absolute pH.     

Spidroin N-terminal domain promotes a pH-dependent association of silk proteins during self-assembly

Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD.     

Anomalous driving force for renal brush border H+/OH-transport characterized by using 6-carboxyfluorescein

The pH, delta pH, and membrane potential dependences of H+/OH-permeability in renal brush border membrane vesicles (BBMV) were studied by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Quantitative H+/OH-fluxes (JH) were obtained from a calibration of the fluorescence response of 6CF to intravesicular pH using vesicles prepared with varying intravesicular and solution pHs. Intravesicular buffer capacity, determined by titration of lysed vesicles, increased monotonically from 140 to 260 mequiv/L in the pH range 5-8. JH was measured by subjecting voltage-clamped BBMV (K+/valinomycin) to preformed pH gradients over the pH range 5-8 and measuring the rate of change of intravesicular pH. For small preformed pH gradients (0.4 pH unit) JH [6 nequiv s-1 (mg of protein)-1] was nearly independent of pH (5-8), predicting a highly pH dependent H+ permeability coefficient. JH increased in a curvilinear manner from 6 to 104 nequiv s-1 (mg of protein)-1 as delta pH increased from 0.4 to 2.5. JH increased linearly [1.6-7.3 nequiv s-1 (mg of protein)-1] with induced K+ diffusion potentials (21-83 mV) in the absence of a pH gradient. These findings cannot be explained by simple diffusion of H+ or OH- or by mobile carrier models. Two mechanisms are proposed, including a lipid diffusion mechanism, facilitated by binding of H+/OH- to fixed sites in the membrane, and a linear H2O strand model, where dissociation of H2O in the membrane fixes H+ and OH- concentrations in strands, which can result in net H+/OH- transport.     

Structural characterization of peptide fragments from hCD81-LEL.

The solution conformation of two peptides [1: PSGSNIISNLFKED; 2: GSSTLTALTTSVLKNNL] from human CD81 (hCD81) large extra-cellular loop (LEL) with known importance in the hepatitis C virus glycoprotein E2 (HCV-E2) binding interaction was characterized using circular dichroism spectroscopy. In addition, the solution structure of peptide 1 that contains a phenylalanine residue (F186 in hCD81) known to be critical in the binding interaction with HCV-E2 was determined using 1D and 2D 1H NMR spectroscopy. Both peptides are unstructured in water but begin forming significant helical conformation following the addition of 20% or more trifluoroethanol (v/v), a result consistent with their alpha-helical conformation found in the native protein. The CD data recorded as a function of pH and NaCl concentration are consistent with stabilization of the helical structure from electrostatic forces for both peptides. Peptide 1 is able to block the binding interaction of recombinant HCV-E2 (rHCV-E2) to hCD81 expressed on Molt-4 T cells at high concentrations (3.5 mM), a low affinity that we attributed to the random coil structure in water.     

Evidence that membrane insertion of the cytosolic domain of Bcl-xL is governed by an electrostatic mechanism

Signals from different cellular networks are integrated at the mitochondria in the regulation of apoptosis. This integration is controlled by the Bcl-2 proteins, many of which change localization from the cytosol to the mitochondrial outer membrane in this regulation. For Bcl-xL, this change in localization reflects the ability to undergo a conformational change from a solution to integral membrane conformation. To characterize this conformational change, structural and thermodynamic measurements were performed in the absence and presence of lipid vesicles with Bcl-xL. A pH-dependent model is proposed for the solution to membrane conformational change that consists of three stable conformations: a solution conformation, a conformation similar to the solution conformation but anchored to the membrane by its C-terminal transmembrane domain, and a membrane conformation that is fully associated with the membrane. This model predicts that the solution to membrane conformational change is independent of the C-terminal transmembrane domain, which is experimentally demonstrated. The conformational change is associated with changes in secondary and, especially, tertiary structure of the protein, as measured by far and near-UV circular dichroism spectroscopy, respectively. Membrane insertion was distinguished from peripheral association with the membrane by quenching of intrinsic tryptophan fluorescence by acrylamide and brominated lipids. For the cytosolic domain, the free energy of insertion (DeltaG degrees x) into lipid vesicles was determined to be -6.5 kcal mol(-1) at pH 4.9 by vesicle binding experiments. To test whether electrostatic interactions were significant to this process, the salt dependence of this conformational change was measured and analyzed in terms of Gouy-Chapman theory to estimate an electrostatic contribution of DeltaG degrees el approximately -2.5 kcal mol(-1) and a non-electrostatic contribution of DeltaG degrees nel approximately -4.0 kcal mol(-1) to the free energy of insertion, DeltaG degrees x. Calcium, which blocks ion channel activity of Bcl-xL, did not affect the solution to membrane conformational change more than predicted by these electrostatic considerations. The lipid cardiolipin, that is enriched at mitochondrial contact sites and reported to be important for the localization of Bcl-2 proteins, did not affect the solution to membrane conformational change of the cytosolic domain, suggesting that this lipid is not involved in the localization of Bcl-xL in vivo. Collectively, these data suggest the solution to membrane conformational change is controlled by an electrostatic mechanism. Given the distinct biological activities of these conformations, the possibility that this conformational change might be a regulatory checkpoint for apoptosis is discussed.     

Na(+)-H+ antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils

Voltage-activated H(+)-selective currents were studied in cultured adult rat alveolar epithelial cells and in human neutrophils using the whole-cell configuration of the patch-clamp technique. The H+ conductance, gH, although highly selective for protons, was modulated by monovalent cations. In Na+ and to a smaller extent in Li+ solutions, H+ currents were depressed substantially and the voltage dependence of activation of the gH shifted to more positive potentials, when compared with the "inert" cation tetramethylammonium (TMA+). The reversal potential of the gH, Vrev, was more positive in Na+ solutions than in inert ion solutions. Amiloride at 100 microM inhibited H+ currents in the presence of all cations studied except Li+ and Na+, in which it increased H+ currents and shifted their voltage-dependence and Vrev to more negative potentials. The more specific Na(+)-H+ exchange inhibitor dimethylamiloride (DMA) at 10 microM similarly reversed most of the suppression of the gH by Na+ and Li+. Neither 500 microM amiloride nor 200 microM DMA added internally via the pipette solution were effective. Distinct inhibition of the gH was observed with 1% [Na+]o, indicating a mechanism with high sensitivity. Finally, the effects of Na+ and their reversal by amiloride were large when the proton gradient was outward (pHo parallel pHi 7 parallel 5.5), smaller when the proton gradient was abolished (pH 7 parallel 7), and absent when the proton gradient was inward (pH 6 parallel 7). We propose that the effects of Na+ and Li+ are due to their transport by the Na(+)-H+ antiporter, which is present in both cell types studied. Electrically silent H+ efflux through the antiporter would increase pHi and possibly decrease local pHo, both of which modulate the gH in a similar manner: reducing the H+ currents at a given potential and shifting their voltage- dependence to more positive potentials. A simple diffusion model suggests that Na(+)-H+ antiport could deplete intracellular protonated buffer to the extent observed. Evidently the Na(+)-H+ antiporter functions in perfused cells, and its operation results in pH changes which can be detected using the gH as a physiological sensor. Thus, the properties of the gH can be exploited to study Na(+)-H+ antiport in single cells under controlled conditions.     

Trifluoroethanol-induced unfolding of concanavalin A: equilibrium and time-resolved optical spectroscopic studies

Conformational structure changes in concanavalin A (Con A), a legume lectin protein which is composed of 18 beta-strands, induced by dissolving in 50% trifluoroethanol (TFE) were monitored at neutral and low pH by far- and near-UV circular dichroism (CD), fluorescence, and FTIR under equilibrium conditions. Stopped-flow studies using CD and fluorescence as well as FTIR, at low and high protein concentration, respectively, were carried out to follow the time-dependent conformation changes occurring after rapid mixing of the protein with TFE. Equilibrium CD results show that, upon addition of TFE, low-concentration Con A transforms to a highly alpha-helical conformation at both neutral and low pH. However, at neutral pH under high protein concentration conditions, aggregation and precipitation are eventually detected with FTIR, indicating that a final beta-structure is attained. Stopped-flow fluorescence shows the existence of an unfolding intermediate for pH 2.0 and 4.5, which could be related to the dissociation of the dimer form. However, evidence for an intermediate is not obtained at pH 6.7, where the native protein is a tetramer. Stopped-flow FTIR is consistent with these results, indicating formation of a H(+)-stabilized intermediate alpha-helical conformation before aggregation develops. Con A in TFE provides an example of an intermediate with non-native secondary structure appearing on the unfolding pathway. On the basis of the kinetic results obtained, an unfolding mechanism is proposed and some stable intermediates are identified. In turn, the slow structural change of Con A induced by TFE provides a useful model system for study of protein unfolding due to its accessibility with several spectroscopic and kinetic tools.     

The interaction of beta-amyloid protein fragment (12-28) with lipid environments.

The neurotoxicity of beta-amyloid protein (beta AP) fragments may be a result of their solution conformation, which is very sensitive to solution conditions. In this work we describe NMR and CD studies of the conformation of beta AP(12-28) in lipid (micelle) environments as a function of pH and lipid type. The interaction of beta AP(12-28) with zwitterionic dodecylphosphocholine (DPC) micelles is weak and alters the conformation when compared to water solution alone. By contrast, the interaction of the peptide with anionic sodium dodecylsulfate (SDS) micelles is strong: beta AP(12-28) is mostly bound, is alpha-helical from K16 to V24, and aggregates slowly. The pH-dependent conformation changes of beta AP(12-28) in solution occur in the pH range at which the side-chain groups of E22, D23, H13, and H14 are deprotonated (pKas ca. 4 and 6.5); the interaction of beta AP(12-28) with SDS micelles alters the pH-dependent conformational transitions of the peptide whereas the weak interaction with DPC micelles causes little change.     

The pH-induced release of iron from transferrin investigated with a continuum electrostatic model.

A reduction in pH induces the release of iron from transferrin in a process that involves a conformational change in the protein from a closed to an open form. Experimental evidence suggests that there must be changes in the protonation states of certain, as yet not clearly identified, residues in the protein accompanying this conformational change. Such changes in protonation states of residues and the consequent changes in electrostatic interactions are assumed to play a large part in the mechanism of release of iron from transferrin. Using the x-ray crystal structures of human ferri- and apo-lactoferrin, we calculated the pKa values of the titratable residues in both the closed (iron-loaded) and open (iron-free) conformations with a continuum electrostatic model. With the knowledge of a residue's pKa value, its most probable protonation state at any specified pH may be determined. The preliminary results presented here are in good agreement with the experimental observation that the binding of ferric iron and the synergistic anion bicarbonate/carbonate results in the release of approximately three H+ ions. It is suggested that the release of these three H+ ions may be accounted for, in most part, by the deprotonation of the bicarbonate and residues Tyr-92, Lys-243, Lys-282, and Lys-285 together with the protonation of residues Asp-217 and Lys-277.     

Interaction of adenylic acid with alkaline earth metal ions in the crystalline solid and aqueous solution. Evidence for the sugar C'2-endo/anti, C'3-endo/anti and C'4-exon/anti conformational changes

The reaction of adenosine 5'-monophosphoric acid (H2-AMP) with the alkaline earth metal ions has been investigated in aqueous solution at neutral pH. The solid salts of Mg-AMP.5H2O, Ca-AMP.6H2O, Sr-AMP.7H2O and Ba-AMP.7H2O were isolated and characterized by Fourier transform infrared, 1H-NMR spectroscopy and X-ray powder diffraction measurements. Spectroscopic and other evidence showed that the Sr-AMP.7H2O and Ba-AMP.7H2O are isomorphous, whereas the Mg-AMP.5H2O and Ca-AMP.6H2O are not similar. The Mg2+ binding is through the N-7 (inner-sphere) and the phosphate group (outer-sphere via H2O), while the Ca2+ binds to the phosphate group (inner-sphere) and to the base N-7 site (outer-sphere through H2O). The Sr2+ and Ba2+ bind to H2O molecules, H-bonding to the N-7, N-1 and the phosphate group (outer-sphere). In aqueous solution, an equilibrium between the inner- and outer-sphere metal ion bindings can be established. The sugar moiety exhibited C'2-endo/anti conformation, in the free H2-AMP acid and the magnesium salt, C'3-endo/anti in the calcium salt and unusual C'4-exo/anti, in the strontium and barium salts.