Dynamics of an integral membrane peptide: a deuterium NMR relaxation study of gramicidin.

Solid state deuterium (2H) NMR inversion-recovery and Jeener-Broekaert relaxation experiments were performed on oriented multilamellar dispersions consisting of 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine and 2H exchange-labeled gramicidin D, at a lipid to protein molar ratio (L/P) of 15:1, in order to study the dynamics of the channel conformation of the peptide in a liquid crystalline phase. Our dynamic model for the whole body motions of the peptide includes diffusion of the peptide around its helix axis and a wobbling diffusion around a second axis perpendicular to the local bilayer normal in a simple Maier-Saupe mean field potential. This anisotropic diffusion is characterized by the correlation times, tau R parallel and tau R perpendicular. Aligning the bilayer normal perpendicular to the magnetic field and graphing the relaxation rate, 1/T1Z, as a function of (1-S2N-2H), where S2N-2H represents the orientational order parameter, wer were able to estimate the correlation time, tau R parallel, for rotational diffusion. Although in the quadrupolar splitting, which varies as (3 cos2 theta D-1), has in general two possible solutions to theta D in the range 0 < or = theta D < or = 90 degrees, the 1/T1Z vs. (1-S2N-2H) curve can be used to determine a single value of theta D in this range. Thus, the 1/T1Z vs. (1-S2N-2H) profile can be used both to define the axial diffusion rate and to remove potential structural ambiguities in the splittings. The T1Z anisotropy permits us to solve for the two correlation times (tau R parallel = 6.8 x 10(-9) s and tau R perpendicular = 6 x 10(-6) s). The simulated parameters were corroborated by a Jeener-Broekaert experiment where the bilayer normal was parallel to the principal magnetic field. At this orientation the ratio, J2(2 omega 0)/J1(omega 0) was obtained in order to estimate the strength of the restoring potential in a model-independent fashion. This measurement yields the rms angle, <theta 2>1/2 (= 16 +/- 2 degrees at 34 degrees C), formed by the peptide helix axis and the average bilayer normal.     

Solvent relaxation in lipid bilayers with dansyl probes

The solvent relaxation properties of the dansyl group attached to two lipids (dansylphosphatidylethanolamine and dansylphosphatidylserine), a fatty acid (dansylundecanoic acid), and two drugs (dansylbenzocaine and dansylpropranolol) were compared in a variety of different lipid systems. Several methods for characterising solvent relaxation were compared in detail for dansylpropranolol in bilayer vesicles of egg phosphatidylcholine. It was shown that the relaxation process is non-monoexponential; nevertheless, for comparative purposes, a model was adopted in which the lifetime associated with the negative exponent in a two exponential decay analysis, obtained at a particular energy on the red edge of emission, was taken as an approximation to a 'solvent relaxation' rate. A negative exponent, indicative of solvent relaxation processes, occurring in the nanosecond time-scale, was found only for dansylpropranolol, dansylPE and dansylundecanoic acid. On addition of the spin probe, 5-doxylstearate, the negative exponent was unaffected in liquid-crystalline phase lipids but was no longer found in gel-phase lipid in the case of dansylpropranolol, while for dansylPE the relaxation time was reduced. On the basis of these types of measurement it was possible to distinguish between different lipid environments using the same probe or between different dansyl environments of the different probes in the same lipid in cases where this would have been difficult or impossible solely on the basis of steady-state or fluorescence lifetime measurements.     

Using nitroxide spin labels. How to obtain T1e from continuous wave electron paramagnetic resonance spectra at all rotational rates.

Historically, the continuous wave electron paramagnetic resonance (CW-EPR) progressive saturation method has been used to obtain information on the spin-lattice relaxation time (T1e) and those processes, such as motion and spin exchange, that occur on a competitive timescale. For example, qualitative information on local dynamics and solvent accessibility of proteins and nucleic acids has been obtained by this method. However, making quantitative estimates of T1e from CW-EPR spectra have been frustrated by a lack of understanding of the role of T1e (and T2e) in the slow-motion regime. Theoretical simulation of the CW-EPR lineshapes in the slow-motion region under increasing power levels has been used in this work to test whether the saturation technique can produce quantitative estimates of the spin-lattice relaxation rates. A method is presented by which the correct T1e may be extracted from an analysis of the power-saturation rollover curve, regardless of the amount of inhomogeneous broadening or the rates of molecular reorientation. The range of motional correlation times from 10 to 200 ns should be optimal for extracting quantitative estimates of T1e values in spin-labeled biomolecules. The progressive-saturation rollover curve method should find wide application in those areas of biophysics where information on molecular interactions and solvent exposure as well as molecular reorientation rates are desired.     

Concerted loop motion triggers induced fit of FepA to ferric enterobactin

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles.     

A molecular model for lipid-protein interaction in membranes: the role of hydrophobic mismatch.

The interaction free energy between a hydrophobic, transmembrane, protein and the surrounding lipid environment is calculated based on a microscopic model for lipid organization. The protein is treated as a rigid hydrophobic solute of thickness dP, embedded in a lipid bilayer of unperturbed thickness doL. The lipid chains in the immediate vicinity of the protein are assumed to adjust their length to that of the protein (e.g., they are stretched when dP > doL) in order to bridge over the lipid-protein hydrophobic mismatch (dP-doL). The bilayer's hydrophobic thickness is assumed to decay exponentially to its asymptotic, unperturbed, value. The lipid deformation free energy is represented as a sum of chain (hydrophobic core) and interfacial (head-group region) contributions. The chain contribution is calculated using a detailed molecular theory of chain packing statistics, which allows the calculation of conformational properties and thermodynamic functions (in a mean-field approximation) of the lipid tails. The tails are treated as single chain amphiphiles, modeled using the rotational isometric state scheme. The interfacial free energy is represented by a phenomenological expression, accounting for the opposing effects of head-group repulsions and hydrocarbon-water surface tension. The lipid deformation free energy delta F is calculated as a function of dP-doL. Most calculations are for C14 amphiphiles which, in the absence of a protein, pack at an average area per head-group ao approximately equal to 32 A2 (doL approximately 24.5 A), corresponding to the fluid state of the membrane. When dP = doL, delta F > 0 and is due entirely to the loss of conformational entropy experienced by the chains around the protein. When dP > doL, the interaction free energy is further increased due to the enhanced stretching of the tails. When dP < doL, chain flexibility (entropy) increases, but this contribution to delta F is overcounted by the increase in the interfacial free energy. Thus, delta F obtains a minimum at dP-doL approximately 0. These qualitative interpretations are supported by detailed numerical calculations of the various contributions to the interaction free energy, and of chain conformational properties. The range of the perturbation of lipid order extends typically over few molecular diameters. A rather detailed comparison of our approach to other models is provided in the discussion.     

Anisotropic 2H-nuclear magnetic resonance spin-lattice relaxation in cerebroside- and phospholipid-cholesterol bilayer membranes.

The axially symmetric powder pattern 2H-nuclear magnetic resonance (NMR) lineshapes observed in the liquid crystalline phase of pure lipid or lipid/cholesterol bilayers are essentially invariant to temperature, or, equivalently, to variations in the correlation times characterizing C-2H bond reorientations. In either of these melted phases, where correlation times for C-2H bond motions are shorter than 10(-7) s, information on the molecular dynamics of the saturated hydrocarbon chain would be difficult to obtain using lineshape analyses alone, and one must resort to other methods, such as the measurement of 2H spin-lattice relaxation rates, in order to obtain dynamic information. In pure lipid bilayers, the full power of the spin-lattice relaxation technique has yet to be realized, since an important piece of information, namely the orientation dependence of the 2H spin-lattice relaxation rates is usually lost due to orientational averaging of T1 by rapid lateral diffusion. Under more favorable circumstances, such as those encountered in the lipid/cholesterol mixtures of this study, the effects of orientational averaging by lateral diffusion are nullified, due to either a marked reduction (by at least an order of magnitude) in the diffusion rate, or a marked increase in the radii of curvature of the liposomes. In either case, the angular dependence of 2H spin-lattice relaxation is accessible to experimental study, and can be used to test models of molecular dynamics in these systems. Simulations of the partially recovered lineshapes indicate that the observed T1 anisotropies are consistent with large amplitude molecular reorientation of the C-2H bond among a finite number of sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Submicrosecond phospholipid dynamics using a long-lived fluorescence emission anisotropy probe.

The use of the long-lived fluorescence probe coronene (mean value of tau(FL) approximately 200 ns) is described for investigating submicrosecond lipid dynamics in DPPC model bilayer systems occurring below the lipid phase transition. Time-resolved fluorescence emission anisotropy decay profiles, measures as a function of increasing temperature toward the lipid-phase transition temperature (T(C)), for coronene-labeled DPPC small unilamellar vesicles (SUVs), are best described in most cases by three rotational decay components (phi(i = 3)). We have interpreted these data using two dynamic lipid bilayer models. In the first, a compartmental model, the long correlation time (phi(N)) is assigned to immobilized coronene molecules located in "gel-like" or highly ordered lipid phases (S-->1) of the bilayer, whereas a second fast rotational time (phi(F) approximately 2-5 ns) is associated with probes residing in more "fluid-like" regions (with corresponding lower ordering, S-->0). Interests here have focused on the origins of an intermediate correlation time (50-100 ns), the associated amplitude (beta(G)) of which increases with increasing temperature. Such behavior suggests a changing rotational environment surrounding the coronene molecules, arising from fluidization of gel lipid. The observed effective correlation time (phi(EFF)) thus reflects a discrete gel-fluid lipid exchange rate (k(FG)). A refinement of the compartmental model invokes a distribution of gel-fluid exchange rates (d(S,T)) corresponding to a distribution of lipid order parameters and is based on an adapted Landau expression for describing "gated" packing fluctuations. A total of seven parameters (five thermodynamic quantities, defined by the free energy versus temperature expansion; one gating parameter (gamma) defining a cooperative "melting" requirement; one limiting diffusion rate (or frequency factor: d(infinity))) suffice to predict complete anisotropy decay curves measured for coronene at several temperatures below the phospholipid T(C). The thermodynamic quantities are associated with the particular lipid of interest (in this case DPPC) and have been determined previously from ultrasound studies, thus representing fixed constants. Hence resolved variables are r(O), temperature-dependent gate parameters (gamma), and limiting diffusion rates (d(infinity)). This alternative distribution model is attractive because it provides a general probe-independent expression for distributed lipid fluctuation-induced probe rotational rates occurring within bilayer membranes below the phospholipid phase transition on the submicrosecond time scale.     

Structure and dynamics of interfacial water in an Lalpha phase lipid bilayer from molecular dynamics simulations

Based on molecular dynamics simulations, an analysis of structure and dynamics is performed on interfacial water at a liquid crystalline dipalmitoylphosphatidycholine/water system. Water properties relevant for understanding NMR relaxation are emphasized. The first and second rank orientational order parameters of the water O-H bonds were calculated, where the second rank order parameter is in agreement with experimental determined quadrupolar splittings. Also, two different interfacial water regions (bound water regions) are revealed with respect to different signs of the second rank order parameter. The water reorientation correlation function reveals a mixture of fast and slow decaying parts. The fast (ps) part of the correlation function is due to local anisotropic water reorientation whereas the much slower part is due to more complicated processes including lateral diffusion along the interface and chemical exchange between free and bound water molecules. The 100-ns-long molecular dynamics simulation at constant pressure (1 atm) and at a temperature of 50 degrees C of 64 lipid molecules and 64 x 23 water molecules lack a slow water reorientation correlation component in the ns time scale. The (2)H(2)O powder spectrum of the dipalmitoylphosphatidycholine/water system is narrow and consequently, the NMR relaxation time T(2) is too short compared to experimental results.     

Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments.

A charge-pulse technique was designed to measure charge movements in the Na-transport mode of the Na,K-ATPase in membrane fragments adsorbed to a planar lipid bilayer with high time resolution. 1) Na+ transport was measured as a function of membrane potential, and 2) voltage-dependent extracellular ion binding and release were analyzed as a function of Na+ concentration and membrane potential. The results could be fitted and explained on the basis of a Post-Albers cycle by simulations with a mathematical model. The minimal reaction sequence explaining the electrogenicity of the pump consists of the following steps: (Na3)E1-P <--> P-E2(Na3) <--> P-E2(Na2) <--> P-E2(Na) <--> P-E2. The conformational change, E1 to E2, is electrogenic (beta 0 < or = 0.1) and the rate-limiting step of forward Na+ transport with a rate constant of 25 s-1 (T = 20 degrees C). The first ion release step, P-E2(Na3) <--> P-E2(Na2), is the major charge translocating process (delta 0 = 0.65). It is probably accompanied by a protein relaxation in which the access structure between aqueous phase and binding site reduces the dielectric distance. The release of the subsequent Na+ ions has a significantly lower dielectric coefficient (delta1 = delta 2 = 0.2). Compared with other partial reactions, the ion release rates are fast (1400 s-1, 700 s-1, and 4000 s-1). On the basis of these findings, a refined electrostatic model of the transport cycle is proposed.     

Dynamics of the phosphate group in phospholipid bilayers. A 31P nuclear relaxation time study.

The spin-lattice relaxation time of the 31P nucleus in the phosphate group of egg yolk phosphatidylcholine multilamellar dispersions has been investigated at four resonant frequencies (38.9, 81.0, 108.9, and 145.7 MHz) in the temperature range from -30 degrees to 60 degrees C. The observed frequency dependence of the relaxation indicates that both dipolar relaxation and relaxation due to anisotropic chemical shielding are significant mechanisms. The experimental data have thus been modeled assuming both mechanisms and the analysis has allowed the contribution of each to the relaxation to be determined along with the correlation time for the molecular reorientation as a function of temperature. Dipolar relaxation was found to dominate at low nuclear magnetic resonance frequencies while at high frequencies the anisotropic chemical shift dominates. The correlation time of the phosphate group is on the order of 10(-9) s at 60 degrees C and increases to approximately 10(-7) s at -30 degrees C. It is observed that the freezing of the buffer which occurs at approximately -8 degrees C has a significant effect on the phosphate group reorientation. This effect of the freezing is to change the activation energy for the phosphate group reorientation from 16.9 KJ/mol above -8 degrees C to 32.5 KJ/mol below -8 degrees C.     

Conformation of tetranactin in solution.

The conformation of tetranactin, an ionophore, in chloroform was investigated by infrared and Raman spectra and by proton and ¹³C magnetic resonances. The infrared spectra show that the structure of its K⁺ complex in the solution is quite similar to that in crystals. The proton spin–spin coupling constants are explained well by assuming that the crystalline structure is retained in solution. The spin–lattice relaxation times of the ¹³C nuclei of the K⁺ complex indicate that its framework is rigid. The correlation time of the overall reorientation of the molecule was calculated to be 9 X 10^?11 sec. On the other hand, the conformation of the complexed form in chloroform differs from that in crystals. Despite the geometrical nonequivalence of the four subunits in the crystalline state, the nuclear magnetic resonance spectra show their magnetic equivalence in the solution. The proton spin–spin coupling constants have values that are averaged by rapid internal rotation. The spin–lattice relaxation times of the ¹³C nuclei in its framework are unexplained by the overall reorientation of the molecule, and reveal the existence of internal motion in the framework. The rate of the local motion of the framework is between 10²–10¹⁰ sec^?1. By comparison of the infrared spectra, it can be said that the mean conformation of the fluctuated framework of the uncomplexed tetranactin in the solution is similar to that of nonactin in the crystalline form, which has an S₄ symmetry axis through the center of the macrocyclic ring.     

The influence of a protein on water dynamics in its vicinity investigated by molecular dynamics simulation

A system containing the globular protein ubiquitin and 4,197 water molecules has been used for the analysis of the influence exerted by a protein on solvent dynamics in its vicinity. Using Voronoi polyhedra, the solvent has been divided into three subsets, i.e., the first and second hydration shell, and the remaining bulk, which is hardly affected by the protein. Translational motion in the first shell is retarded by a factor of 3 in comparison to bulk. Several molecules in the first shell do not reach the diffusive regime within 100 ps. Shell-averaged orientational autocorrelation functions, which are also subject to a retardation effect, cannot be modeled by a single exponential time law, but are instead well-described by a Kohlrausch-Williams-Watts (KWW) function. The underlying distribution of single-molecule rotational correlation times is both obtained directly from the simulation and derived theoretically. The temperature dependence of reorientation is characterized by a strongly varying correlation time, but a virtually temperature-independent KWW exponent. Thus, the coupling of water structure relaxation with the respective environment, which is characteristic of each solvation shell, is hardly affected by temperature. In other words, the functional form of the distributions of single-molecule rotational correlation times is not subject to a temperature effect. On average, a correlation between reorientation and lifetimes of neighborhood relations is observed. © 1996 Wiley-Liss, Inc.     

Dielectric relaxation in hydroxyethyl cellulose

This paper presents the results of the dielectric analysis of hydroxyethyl cellulose (HEC) in the temperature range 100–350 K and in the frequency range 20 Hz–1 MHz. The results show a distinct broad relaxation process in the temperature range 150–250 K with activation energy of about 33.4 MJ/kmol. The strength of the relaxation, ε_s−ε∞, increases only slightly with temperature and the apparent increase of the height of the relaxation maximum is attributed to an increase of the co-operativity parameter . This increase of is interpreted as decrease of the co-operativity of dipole motions with increasing temperature. The origin of the relaxation is ascribed to the reorientation of double ethylene oxide groups or part of these.     

Molecular response of the lipid headgroup to bilayer hydration monitored by 2H-NMR.

The effect of hydration on the conformation and dynamics of the phosphatidylcholine headgroup has been investigated by 2H-NMR measurements of liquid crystalline dioleoylphosphatidylcholine in multilamellar liposomes. Deuterium quadrupole splittings (delta nu Q) and spin-lattice relaxation rates (1/T1) were recorded for three selectively labeled headgroup segments (alpha, beta, and gamma) over the range of water/lipid mole ratios from 4 to 100. The smooth changes in delta nu Q and 1/T1 are found to essentially parallel each other and can be described by a single exponential decay function. Progressive hydration thus induces a concerted change in headgroup conformation together with an increase in its rate of motion (detected by delta nu Q and 1/T1, respectively). The enhanced mobility is partially due to a shift in the lipid phase transition temperature (as monitored by differential scanning calorimetry) and is furthermore attributed to an entropic contribution. It is concluded that the choline dipole becomes slightly raised in its average orientation into the aqueous layer and that the rate is increased at which the headgroup is fluctuating and protruding. The observed molecular changes can thus be accommodated within a model where the effective accessible headgroup volume expands with increasing hydration.     

Interactions of the apolipoprotein A5 gene polymorphisms and alcohol consumption on serum lipid levels

Background

Little is known about the interactions of apolipoprotein (Apo) A5 gene polymorphisms and alcohol consumption on serum lipid profiles. The present study was undertaken to detect the interactions of ApoA5–1131T>C, c.553G>T and c.457G>A polymorphisms and alcohol consumption on serum lipid levels.

Methodology/Principal Findings

A total of 516 nondrinkers and 514 drinkers were randomly selected from our previous stratified randomized cluster samples. Genotyping was performed by polymerase chain reaction and restriction fragment length polymorphism. The levels of serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), ApoA1 and ApoB were higher in drinkers than in nondrinkers (P<0.05–0.001). The genotypic and allelic frequencies of three loci were not different between the two groups. The interactions between –1131T>C genotypes and alcohol consumption on ApoB levels (P<0.05) and the ApoA1/ApoB ratio (P<0.01), between c.553G>T genotypes and alcohol consumption on low-density lipoprotein cholesterol (LDL-C) levels (P<0.05) and the ApoA1/ApoB ratio (P<0.05), and between c.457G>A genotypes and alcohol consumption on TG levels (P<0.001) were detected by factorial regression analysis after controlling for potential confounders. Four haplotypes (T-G-G, C-G-G, T-A-G and C-G-T) had frequencies ranging from 0.06 to 0.87. Three haplotypes (C-G-G, T-A-G, and C-G-T) were significantly associated with serum lipid parameters. The –1131T>C genotypes were correlated with TG, and c.553G>T and c.457G>A genotypes were associated with HDL-C levels in nondrinkers (P<0.05 for all). For drinkers, the –1131T>C genotypes were correlated with TC, TG, LDL-C, ApoB levels and the ApoA1/ApoB ratio (P<0.01 for all); c.553G>T genotypes were correlated with TC, TG, HDL-C and LDL-C levels (P<0.05–0.01); and c.457G>A genotypes were associated with TG, LDL-C, ApoA1 and ApoB levels (P<0.05–0.01).

Conclusions

The differences in some serum lipid parameters between the drinkers and nondrinkers might partly result from different interactions of the ApoA5 gene polymorphisms and alcohol consumption.     

Proton NMR T1, T2, and T1 rho relaxation studies of native and reconstituted sarcoplasmic reticulum and phospholipid vesicles.

The phospholipids protons of native and reconstituted sarcoplasmic reticulum (SR) membrane vesicles yield well-resolved nuclear magnetic resonance (NMR) spectra. Resonance area measurements, guided by the line shape theory of Bloom and co-workers, imply that we are observing a large fraction of the lipid intensity and that the protein does not appear to reduce the percent of the signal that is well resolved. We have measured the spin-lattice (T1) and spin-spin (T2) relaxation rates of the choline, methylene, and terminal methyl protons at 360 MHz and the spin-lattice relaxation rate in the rotating frame (T1 rho) at 100 MHz. Both the T1 and T2 relaxation rates are single exponential processes for all of the resonances if the residual water proton signal is thoroughly eliminated by selective saturation. The T1 and T2 relaxation rates increase as the protein concentration increases, and T2 rate decrease with increasing temperature. This implies that the protein is reducing both high frequency (e.g., trans-gauche methylene isomerizations) and low frequency (e.g., large amplitude, chain wagging) lipid motions, from the center of the bilayer to the surface. It is possible that spin diffusion contributes to the effect of protein on lipid T1's although some of the protein-induced T1 change is due to motional effects. The T2 relaxation times are observed to be near 1 ms for the membranes with highest protein concentration and approximately 10 ms for the lipids devoid of protein. This result, combined with the observation that the T2 rates are monophasic, suggests that at least two lipid environments exist in the presence of protein, and that the lipids are exchanging between these environments at a rate greater than 1/T2 or 10(3) s-1. The choline resonance yields single exponential T1 rho relaxation in the presence and absence of protein, whereas the other resonances measured exhibit biexponential relaxation. Protein significantly increases the single T1 rho relaxation rate of the choline peak while primarily increasing the T1 rho relaxation rate of the more slowly relaxing component of the methylene and methyl resonances.     

Effect of sodium chloride on a lipid bilayer

Electrostatic interactions govern structural and dynamical properties of membranes and can vary considerably with the composition of the aqueous buffer. We studied the influence of sodium chloride on a pure POPC lipid bilayer by fluorescence correlation spectroscopy experiments and molecular dynamics simulations. Increasing sodium chloride concentration was found to decrease the self-diffusion of POPC lipids within the bilayer. Self-diffusion coefficients calculated from the 100 ns simulations agree with those measured on a millisecond timescale, suggesting that most of the relaxation processes relevant for lipid diffusion are faster than the simulation timescale. As the dominant effect, the molecular dynamics simulations revealed a tight binding of sodium ions to the carbonyl oxygens of on average three lipids leading to larger complexes with reduced mobility. Additionally, the bilayer thickens by approximately 2 A, which increases the order parameter of the fatty acyl chains. Sodium binding alters the electrostatic potential, which is largely compensated by a changed polarization of the aqueous medium and a lipid dipole reorientation.     

Cumulative viral load and virologic decay patterns after antiretroviral therapy in HIV-infected subjects influence CD4 recovery and AIDS

Background

The impact of viral load (VL) decay and cumulative VL on CD4 recovery and AIDS after highly-active antiretroviral therapy (HAART) is unknown.

Methods and Findings

Three virologic kinetic parameters (first year and overall exponential VL decay constants, and first year VL slope) and cumulative VL during HAART were estimated for 2,278 patients who initiated HAART in the U.S. Military HIV Natural History Study. CD4 and VL trajectories were computed using linear and nonlinear Generalized Estimating Equations models. Multivariate Poisson and linear regression models were used to determine associations of VL parameters with CD4 recovery, adjusted for factors known to correlate with immune recovery. Cumulative VL higher than the sample median was independently associated with an increased risk of AIDS (relative risk 2.38, 95% confidence interval 1.56–3.62, p<0.001). Among patients with VL suppression, first year VL decay and slope were independent predictors of early CD4 recovery (p = 0.001) and overall gain (p<0.05). Despite VL suppression, those with slow decay during the first year of HAART as well as during the entire therapy period (overall), in general, gained less CD4 cells compared to the other subjects (133 vs. 195.4 cells/µL; p = 0.001) even after adjusting for potential confounders.

Conclusions

In a cohort with free access to healthcare, independent of established predictors of AIDS and CD4 recovery during HAART, cumulative VL and virologic decay patterns were associated with AIDS and distinct aspects of CD4 reconstitution.     

Proton NMR spin grouping and exchange in dentin.

The nuclear magnetic resonance spin-grouping technique has been applied to dentin from human donors of different ages. The apparent T2, T1, and T1 rho have been determined for natural dentin, for dentin which has been dried in vacuum, and for dried dentin which has been rehydrated in an atmosphere with 75% relative humidity. All apparent spin relaxation has been analyzed for exchange between the spin groups in which the dentin protons exist; the analyses incorporate the results of selective inversion recovery T1 measurements which better probe the effects of exchange. The exchange analyses of the high fields and rotating frame spin-lattice relaxation have also been correlated to determine uniquely the inherent relaxation parameters of the proton spin groups constituting the dentin magnetization. The natural dentin contains protons on water, protein, and hydroxy apatite; these spins contribute 50%, 45%, and 5% to the total dentin proton magnetization, respectively. The water exists in three distinct environments, the dynamics of each environment has been modeled. In the natural dentin 30% of the water undergoes uni-axial reorientation. 52% of the water has similar relaxation characteristics to bound water hydrating a large molecule, and the majority of the remaining water acts as bulk water undergoing isotropic reorientation. The results are independent of the age of the donor.     

Thermal equilibration between the M and N intermediates in the photocycle of bacteriorhodopsin.

The stages in the photocycle of bacteriorhodopsin (BR) involving the M and N intermediates are investigated using a double pulse excitation method. A first (cycling) pulse at 532 nm is followed, with an appropriate time delay, by a second pulse (337, 406, 446, or 470 nm) which induces the M-->BR back-photoreaction. After depletion by the second pulse a repopulation of M in the millisecond range is observed which is interpreted in terms of a thermal N-->M relaxation. It is thus concluded that a (thermal) M<-->N equilibrium accounts for the biphasic decay of M in the BR photocycle. Other models for this stage of the light-driven proton-pump are therefore unnecessary.