Determination of the complete order parameter tensor for a lipid methylene group from 1H- and 2H-NMR spin labels

Proton-proton dipolar splittings are obtained as a function of temperature for the α-methylene in a potassium palmitate - (β-ω) - d₂₉ (70 wt.%) / D₂O (30 wt.%) sample above and below the gel to liquid crystal phase transition. These splittings and corresponding deuteron quadrupole splittings are used to specify the complete order parameter tensor for the α-methylene group. Deduction of lipid structural information from the complete-order parameter tensor is discussed.     

The temperature dependence of water and counter ion order in soap-water mesophases. A deuterium and sodium NMR study

Deuterium magnetic resonance (DMR) spectra of the water and hydrocarbon chains in potassium and sodium palmitates and sodium magnetic resonance spectra of sodium in sodium palmitate demonstrate correlations between water, hydrocarbon chain and counter ion order. In the lamellar phase of potassium palmitate the order parameters inferred from DMR splittings of D₂O and the first few methylene chain segments initially increase and then decrease with increasing temperature. This is explained in terms of a model where the lipid-water structure at low temperatures imposes a direction for which all the order parameters are smaller than for the higher temperature structure for purely geometric reasons. As temperature increases the structuring effect of water decreases and there is an “apparent” increase in order until at even higher temperatures there is an intrinsic decrease in order parameter. In addition, for potassium palmitate the DMR splittings of D₂O and the first few methylene segments indicate a “phase transition” within the liquid crystalline phase.     

Inelastic neutron scattering investigations of molecular nanomagnets

Inelastic neutron scattering, probing the temporal spin–spin correlation at the microscopic scale, is a powerful technique to study the magnetic behaviour of molecular nanomagnets. Experiments at different energy scales and different energy-transfer resolution allow precise determinations of the parameters defining the effective Hamiltonians used to model the diverse physical properties exhibited by this class of materials. The intrinsic disadvantage of the technique (low flux, requiring a sample mass in the gram scale) is over-compensated by the large amount of information that can be straightforwardly extracted. Zero-field splittings and exchange interactions can be determined with a large degree of confidence, shedding light on important issues such as magnetization tunnelling in giant-spin clusters, or the occurrence of quantum coherence phenomena and their consequences on macroscopic observables.     

Direct observation of the properties of cholesterol in membranes by deuterium NMR

The properties of cholesterol in bilayers of egg phosphatidylcholine (PC) were investigated directly by means of ²H-NMR of specifically-deuterated species (C3, C7, C26, C27). Quadrupole splittings were a measure of molecular ordering, and relaxation times T₁ and T_2e were indicators of rates of motion. The importance of the use of echoes for spectral acquisition is emphasised, particularly to obtain accurate values of the quadrupole splitting. In the case of overlapping powder patterns from two labelled positions, the use of the absolute value mode of spectral presentation is shown to yield reasonable estimates of the individual quadrupole splittings. Spectral properties were monitored as a function of cholesterol concentration and temperature. Increasing cholesterol concentration led to a high degree of ordering for the rigid ring system of cholesterol, approaching a molecular order parameter of 0.8 at 50 mol% cholesterol. The isopropyl methyl groups were in all cases less ordered anmore mobile than the ring system, but responded in a similar fashion to variable cholesterol concentration and temperature. The observation of a minimum in the temperature dependence of T₁ for cholesterol-7,7-d₂ led to a direct estimate of its correlation time for molecular motion, 3.5 × 10^?9 s rad^?1. This indicates that the overall rate of motion of cholesterol is considerably slower than that of the lipids in which it is located. The short T_2e values suggest that the motional spectrum of cholesterol is rich in low frequencies. The parallel temperature and cholesterol dependences of quadrupole splittings for different positions on the rigid ring system of cholesterol indicate that the position of the axis of motional averaging of the molecule is not changing, and is the same as that determined in an earlier study. It is emphasised that the steep temperature dependence and small quadrupole splittings for the chain isopropyl methyl groups of cholesterol do not necessarily indicate a high degree of disorder, but may be due to their axes of motional averaging lying at angles close to 54° with respect to the director of the ordered lipids.     

The synthesis of amino acids and sugars on an inorganic template from constituents of the prebiotic atmosphere

Inelastic Electron Tunnelling Spectroscopy (IETS) has been used to identify the reaction products present on an alumina surface when it is exposed to likely components of the earth's prebiotic atmosphere. The alumina barrier of Al-AlO _x -Pb tunnelling junctions have been exposed to water; aqueous ammonia; wet carbon monoxide gas and to aqueous formaldehyde vapour under normal atmospheric conditions at room temperature. The water spectrum shows strong coincidence with that of a genuine sample of formic acid. It is proposed that atmospheric CO₂ is involved in this surface catalyzed reaction. The aqueous ammonia spectrum is assigned as an amino acid species produced from ammonia, water and atmospheric carbon dioxide. This spectrum compares very closely with the tunnelling spectrum of a genuine sample of glycine. The wet carbon monoxide spectrum and the aqueous formaldehyde spectrum have been produced by an infusion doping process. These spectra of CO and aqueous formaldehyde are assigned as a sugar like polymer or a sugar formed on the alumina surface. A tunnelling spectrum of D(–) fructose has been produced to aid this assignment. The role of an inorganic template such as alumina in the original prebiotic synthesis of amino acids and sugars is considered.     

Calculations of quantum tunnelling between closed and open states of sodium channels

Transitions between states of ion channels have previously been considered in terms of classical statistical mechanics. However, transitions in many systems, including some organic molecules, are known to occur by quantum mechanical tunnelling. In this report, we have calculated the time for sodium channel activation by tunnelling, starting from a mechanistic model based on the structural models of Catterall and Guy. In doing this, we have calculated the Coulomb interactions between the S4 -helix and negative residues on nearest-neighbor helices and have included longer range interactions in terms of an effective background interaction. Periodic pairing of charges between the S4 and adjacent helices in the model causes the resting and depolarized states of the channel to correspond to local minima in the S4 potential energy curve. Harmonic potentials closely fit the energy curves around each of the two minima and the energy barrier between them is closely modelled by a parabola. These approximations allow a semiclassical calculation of the S4 helix's tunnelling rate to be made. At 37°C, for an interhelix axial spacing of 10 Å, tunnelling times in the range of 1 s to a few ms were computed for a single S4 segment, depending of the equilibrium temperature of the cell membrane.     

X-ray magnetic circular dichroism investigation of the superparamagnetic transition-metal ion-cluster r-Mn12Bz

We present the results of an X-ray magnetic circular dichroism investigation of a reduced version of Mn₁₂ benzoate. At variance with the parent Mn₁₂ benzoate compound, which, analogously to Mn₁₂ acetate, has a ground-state spin equal to ten, the reduced species has a ground-state with total spin S = 19/2. The half-integer spin in the ground-state makes this compound an appealing system where to test parity effects on the efficiency of the quantum tunnelling of the magnetisation. We exploited the sensitivity of X-ray magnetic circular dichroism to the oxidation state of the absorbing metal ion to obtain information about the internal structure of the reduced Mn₁₂ benzoate. In particular, we performed multiplet calculations to analyse the X-ray magnetic circular dichroism spectra at the manganese L_2,3 edge and identify the contribution of the Mn^II ion resulting by the reduction process.     

Assignment of Proton Endor Resonances of Nitroxyl Spin-Labels in Frozen Solution

Spin-label nitroxyl derivatives of tetramethylpyrroline and tetramethylpyrrolidine in frozen solutions of perdeuterated methanol have been characterized by electron nucleus double resonance (ENDOR spec-troscopy). With use of selectively deuterated derivatives of 2,2,5,5-tetramethylpyrroline-l-oxyl-3-carboxamide, proton ENDOR resonance features have been assigned to the vinylic proton in the five membered pyrrolinyl ring and to the methyl groups. The ENDOR resonance features were analyzed on the basis of their dependence on H₀. Two pairs of resonance features were assigned to the vinylic proton and were shown to correspond to ‖ and ⊥ hyperfine coupling (hfc) components. Six pairs of resonance features were ascribed to the methyl groups. The proton ENDOR spectra of the 3-carboxylic acid spin-label derivatives of tetramethylpyrroline and of tetramethylpyrrolidine compounds exhibited comparable features with nearly identical line splittings. From the observed ENDOR splittings, we have estimated the isotropic hfc component of the vinylic proton in 2,2,5,5-tetramethylpyrroline-l-oxyl-3-carboxamide to be -1.81 ± 0.04 MHz in frozen methanol. On the basis of the anisotropic dipolar hfc components, the electron-to-vinylic proton distance is estimated as 3.78 ± 0.01 Å. in excellent agreement with that of 3.79 Å calculated from X-ray defined coordinates.     

15N nmr spectroscopy. I. Polysarcosine and related sequence polypeptides

The natural abundance ¹⁵N nmr spectra of linear polysarcosine (DP = 35) has been recorded in Me₂SO and H₂O solution. Because of cis/trans isomerization at the peptide bond, a broad signal with several splittings was observed. These splittings appear to reflect the influence of three peptide bonds on a single N atom. The ¹⁵N signals from the sequence polypeptides (β-Ala-Sar-Gly)_n and (β-Ala-Sar-D ,L -Ala)_n also show a cis/trans splitting, as well as chemical shifts which are dependent on the peptide sequence. The tertiary nitrogen of the sarcosyl residue has a T₁ relaxation time which is longer than the T₁ for secondary nitrogens of the other amino acids. The nuclear Overhauser effect is also discussed.     

Exploring biomolecular machines: energy landscape control of biological reactions

For almost 15 years, our Pathway model has been the most powerful model in terms of predicting the tunnelling mechanism for electron transfer (ET) in biological systems, particularly proteins. Going beyond the conventional Pathway models, we have generalized our method to understand how protein dynamics modulate not only the Franck-Condon factor, but also the tunnelling matrix element. We have demonstrated that when interference among pathways modulates the electron tunnelling interactions in proteins (particularly destructive interference), dynamical effects are of critical importance. Tunnelling can be controlled by protein conformations that lie far from equilibrium-those that minimize the effect of destructive interference during tunnelling, for example. In the opposite regime, electron tunnelling is mediated by one (or a few) constructively interfering pathway tubes and dynamical effects are modest. This new mechanism for dynamical modulation of the ET rate has been able to explain and/or predict several rates that were later confirmed by experiment. However, thermal fluctuations can also affect these molecular machines in many other ways. For example, we show how global transformations, which control protein functions such as allostery, may involve large-scale motion and possibly partial unfolding during the reaction event.     

KCTCCA,a peptide-based facilitator for bioelectrochemistry

Electrochemical and scanning tunnelling microscopy (STM) studies have been carried out to investigate the suitability of the hexapeptide KCTCCA as a facilitator for bioelectrochemistry. The stable, quasi-reversible electro-chemical response of cytochromeb ₅₆₂ on a KCTCCA modified gold electrode and the high degree of surface coverage of KCTCCA on gold (111), as observed by STM, indicate applicability of the molecule as an electro-chemical facilitator.     

Aggregated Multicast – A Comparative Study

Though IP multicast is resource efficient in delivering data to a group of members simultaneously, it suffers from scalability problem with the number of concurrently active multicast groups because it requires a router to keep forwarding state for every multicast tree passing through it. To solve this state scalability problem, we proposed a scheme, called aggregated multicast. The key idea is that multiple groups are forced to share a single delivery tree. In our earlier work, we introduced the basic concept of aggregated multicast and presented some initial results to show that multicast state can be reduced. In this paper, we develop a more quantitative assessment of the cost/benefit trade-offs. We propose an algorithm to assign multicast groups to delivery trees with controllable cost and introduce metrics to measure multicast state and tree management overhead for multicast schemes. We then compare aggregated multicast with conventional multicast schemes, such as source specific tree scheme and shared tree scheme. Our extensive simulations show that aggregated multicast can achieve significant routing state and tree management overhead reduction while containing the expense of extra resources (bandwidth waste and tunnelling overhead). We conclude that aggregated multicast is a very cost-effective and promising direction for scalable transit domain multicast provisioning.     

Measurement of dipolar couplings in a transducin peptide fragment weakly bound to oriented photo-activated rhodopsin

Rhodopsin-containing disks, isolated from rod outer segments of bovine retina, align at high magnetic fields with their membrane normal parallel to the magnetic field. After light-activation of rhodopsin, transient binding of the C-terminal transducin undecapeptide, selectively labeled with ¹⁵N at Leu⁵ and Gly⁹, results in residual dipolar contributions to the ¹J_NH splittings for these two residues. Both residues show ¹J_NH splittings which are smaller than in the dark-adapted or rhodopsin-free sample, and return to their isotropic values at a rate determined by the decay of the meta II state of rhodopsin. The dipolar couplings indicate that in the bound state, N-H vectors of Leu⁵ and Gly⁹ make angles of 48±4° and 40±8°, respectively, with the disk normal. These `transferred' dipolar couplings potentially offer a useful method for studying the conformation and orientation of flexible, low affinity ligands when bound to oriented integral membrane receptors.     

Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling

Early studies of enzyme-catalysed hydride transfer reactions indicated kinetic anomalies that were initially interpreted in the context of a 'tunnelling correction'. An alternate model for tunnelling emerged following studies of the hydrogen atom transfer catalysed by the enzyme soybean lipoxygenase. This invokes full tunnelling of all isotopes of hydrogen, with reaction barriers reflecting the heavy atom, environmental reorganization terms. Using the latter approach, we offer an integration of the aggregate data implicating hydrogen tunnelling in enzymes (i.e. deviations from Swain-Schaad relationships and the semi-classical temperature dependence of the hydrogen isotope effect). The impact of site-specific mutations of enzymes plays a critical role in our understanding of the factors that control tunnelling in enzyme reactions.     

The Role of Colony Size on Tunnel Branching Morphogenesis in Ant Nests

Many ant species excavate nests that are made up of chambers and interconnecting tunnels. There is a general trend of an increase in nest complexity with increasing population size. This complexity reflects a higher ramification and anastomosis of tunnels that can be estimated by the meshedness coefficient of the tunnelling networks. It has long been observed that meshedness increases with colony size within and across species, but no explanation has been provided so far. Since colony size is a strong factor controlling collective digging, a high value of the meshedness could simply be a side effect of a larger number of workers. To test this hypothesis, we study the digging dynamics in different group size of ants Messor sancta. We build a model of collective digging that is calibrated from the experimental data. Model''s predictions successfully reproduce the topological properties of tunnelling networks observed in experiments, including the increase of the meshedness with group size. We then use the model to investigate situations in which collective digging progresses outward from a centre corresponding to the way tunnelling behaviour occurs in field conditions. Our model predicts that, when all other parameters are kept constant, an increase of the number of workers leads to a higher value of the meshedness and a transition from tree-like structures to highly meshed networks. Therefore we conclude that colony size is a key factor determining tunnelling network complexity in ant colonies.     

Phospholipid hydration studied by deuteron magnetic resonace spectroscopy

Deuteron magnetic resonance spectra were obtained from ²H₂O in mixtures with egg lecithin, egg phosphatidylethanolamine, and ox brain sodium phosphatidylserine. The acid form of phosphatidylserine does not hydrate. Details of the different hydration “shells” were obtained by studying the spectral splittings as a function of ²H₂O concentration. Several different types of water are present, including bulk water (exchanging only slowly with water associated with the lipid), “trapped” water (not present with phosphatidylethanolamine), and up to three types of bound water. The spectral splittings characteristic of each water environment yielded information about the water binding energies and degrees of anisotropy of motion of the phospholipid polar groups; lecithin polar groups have least motional restriction and sodium phosphatidylserine most, while phosphatidylethanolamine binds water most tightly.Spectra of some lecithin and phosphatidylserine dispersions varied with time, due to a slow reorganization of randomly oriented multilamellar regions into longer, more ordered systems, with a length of about 1 μm. At ?20°C the timescales of the change were of the order of a week and a month for lecithin and phosphatidylserine respectively.Complex changes in the spectra were observed as the temperature was raised; these are interpreted in terms of changes in the motions of the phospholipid molecules.     

The mm-wave rotational spectrum of dichlorodimethylgermane

The rotational spectra of three isotopologues of dichlorodimethylgermane were measured by free jet absorption millimeterwave spectroscopy. A partial r₀ structure was obtained. The barrier to internal rotation of the methyl groups is too high to cause splittings observable within our spectral resolution. Corresponding to the resolving power of the experiment, the lower limit to the V₃ barrier is about 5.8 kJ/mol.     

Generalized pattern search algorithm for Peptide structure prediction

Finding the near-native structure of a protein is one of the most important open problems in structural biology and biological physics. The problem becomes dramatically more difficult when a given protein has no regular secondary structure or it does not show a fold similar to structures already known. This situation occurs frequently when we need to predict the tertiary structure of small molecules, called peptides. In this research work, we propose a new ab initio algorithm, the generalized pattern search algorithm, based on the well-known class of Search-and-Poll algorithms. We performed an extensive set of simulations over a well-known set of 44 peptides to investigate the robustness and reliability of the proposed algorithm, and we compared the peptide conformation with a state-of-the-art algorithm for peptide structure prediction known as PEPstr. In particular, we tested the algorithm on the instances proposed by the originators of PEPstr, to validate the proposed algorithm; the experimental results confirm that the generalized pattern search algorithm outperforms PEPstr by 21.17% in terms of average root mean-square deviation, RMSD C_α.     

The molecular dynamics of cholesterol in bilayer membranes: A deuterium NMR study

The ²H-NMR spectra of selectively deuterated cholesterol, intercalated in egg phosphatidyl-choline, were examined. The orientation of the axis of motional averaging was calculated using the observed quadrupole splittings and the atomic coordinates. With the known orientation of the rotation axis, quadrupole splittings observed for deuterium labels on cholesterol can be related to the molecular order parameter of the sterol. In addition, knowledge of the axis orientation allows prediction of the magnitudes of quadrupole splittings for deuterium at other positions, which is useful in the choice of labelling for particular applications. Finally, preliminary relaxation time measurements yield information on the rates of anisotropic motion of cholesterol in bilayer membranes.     

2H nuclear magnetic resonance of the gramicidin A backbone in a phospholipid bilayer.

Solid-state 2H NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44 degrees C and 53 degrees C, from gramicidin A labels in which the proton attached to the alpha carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-2H bonds. The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, (delta vQL/delta vQD), is expected to be 0.97 +/- 0.04 for a relaxed right-handed beta 6.3LD helix, while a ratio of 0.904 +/- 0.003 is expected for a left-handed beta LD6.3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta LD6.3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta LD6.3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with water) with greater flexibility would show additional motional narrowing for labels located there.