Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: Effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK_a values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CγH, whereas the deviation of pK_a from the reference value for Glu4 and Glu8 CγH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Kinetics of channelized membrane ions in magnetic fields

The cyclotron resonance model for channel ion transport in weak magnetic fields is extended to include damping losses. The conductivity tensor is obtained for different electric field configurations, including the circuital field E phi normal to the channel axis. The conductivity behavior close to the cyclotron resonance frequency omega c is compared to existing Ca2+-efflux data in the literature. A collision time of .023 s results from this comparison under the assumption that K+ ions are transiting in a 0.35 G field. We estimate a mean kinetic energy of 3.5 eV for this ion at resonance. This model leads to discrete modes of vibration (eigenfrequencies) in the ion-lattice interaction, such that omega n = n omega c. The presence of such harmonics is compatible with recent results by Blackman et al. [1985b] and McLeod et al. [1986] with the interesting exception that even modes do not appear in their observations, whereas the present model has no restriction on n. This harmonic formalism is also consistent with another reported phenomenon, that of quantized multiple conductances in single patch-clamped channels.     

Measurement of Free Intracellular Calcium in the Brain by 19F-Nuclear Magnetic Resonance Spectroscopy

We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F-nuclear magnetic resonance calcium chelator, 5,5'-F2-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.     

Phosphorus-31 magnetic resonance spectroscopy of forearm flexor muscles in student rowers using an exercise protocol adjusted for differences in cross-sectional muscle area

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J.cm-2 CSAmax for 1 min and an increment of 1 J.cm-2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of 31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2, P less than 0.05]. The absolute maximal exercise intensity (J.min-1) was greater in the rowers than in the control subjects. However, the maximal exercise intensity per unit of muscle cross sectional area (J.min-1.cm-2) was not significantly different between the two groups. During mild to moderate exercise intensities, a decrease in phosphocreatine and an increase in inorganic phosphate before the onset of acidosis were significantly less in the rowers, indicating a requirement of less adenosine 5'-diphosphate to drive adenosine 5'-triphosphate production. The onset of acidosis was also significantly delayed in the rowers. No difference was observed in forearm blood flow between the two groups at the same exercise intensity (J.min-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformations and dynamics of surfactants in micelles and liquid crystals by nuclear magnetic relaxation

The order parameters as well as the rates of overall and internal motions of aggregated surfactants can be obtained from deuteron and carbon-13 nuclear relaxation experiments. The main contribution to the relaxation is generally the quadrupolar coupling (²H) or the short range dipolar interaction with protons (¹³C). In some cases it is convenient to derive the same information from the¹³C relaxation induced by long range dipolar interactions with a paramagnetic probe exchanging rapidly among the polar heads of surfactant molecules. This paper outlines the methods of interpretation of relaxation data by means of a rotational jump model of internal motions, taking into account most of the accessible conformers. The conformational and dynamical parameters are obtained from the magnetic field dependence of the longitudinal relaxation rates (micelles) or from the simultaneous fit of these rates and of the dipolar or quadrupolar splittings (liquid crystals). Some examples of application of these methods are given from recent works on single and double detailed surfactants.