Slow motions in lipid bilayers. Direct detection by two-dimensional solid-state deuterium nuclear magnetic resonance.

Two-dimensional solid-state 2H NMR spectroscopy of specifically deuteriated lipids is used to detect and to characterize the rate and mode of slow motions in two lipid bilayer systems. Lateral diffusion of lipid molecules over the curved surface of dipalmitoylphosphatidylcholine liposomes can be detected by two-dimensional exchange 2H NMR and it is shown that molecular orientational exchange is complete on the timescale of 100 ms. In contrast, it is shown that for the glycolipid 1,2-di-O-tetradecyl-3-O-Beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL), there is no evidence of a corresponding orientational exchange in the liquid-crystalline phase suggesting that this lipid forms relatively flat bilayers. In the gel phase of hydrated multibilayers of beta-DTGL, a slow (10(3) s(-1)) whole molecule axial motion is demonstrated at 40 degrees C. Comparison of the experimental and simulated 2D-NMR ridge patterns suggests that large angle jumps about the long molecular axis, rather than small step Brownian diffusion, can best account for the 2D-exchange spectra of beta-DTGL in the gel phase. The significance of this technique for the study of dynamics in other biological systems is discussed.     

Packing of cholesterol molecules in human low-density lipoprotein

High-resolution, proton-decoupled 13C nuclear magnetic resonance spectra (90.55 MHz) of human low-density lipoprotein (LDL) have been employed to investigate the physical state of unesterified cholesterol molecules in this particle. Approximately half of the cholesterol molecules in LDL were replaced with [4-13C]cholesterol by exchange from Celite. About two-thirds of the cholesterol molecules contribute to a resonance at delta 41.8 from the C-4 atom. This signal is assigned to cholesterol molecules located at the surface of the LDL particle in a mixed monolayer with phospholipid molecules; the spin-lattice relaxation of the C-4 nucleus of such cholesterol molecules is enhanced by the presence of Mn2+ ions in the aqueous phase. The remaining one-third of the cholesterol molecules are apparently neither associated with phospholipid nor exposed to the aqueous phase; these cholesterol molecules are presumed to be located in the core of the particle. Cholesterol molecules in the two microenvironments are in slow exchange on the NMR time scale but in fast exchange on a biological time scale, so that the cholesterol molecules in LDL behave physiologically as one pool. There is a loss of about 20% of the intensity of the N(CH3)3 resonance from phosphatidylcholine and sphingomyelin molecules in the LDL spectrum; this is attributed to the presence of apolipoprotein B in the surface of LDL particles, which may immobilize some of the phospholipid polar groups. Spin-lattice relaxation time measurements suggest that the fast axial motions of cholesterol molecules in the surface of LDL are the same as in high-density lipoprotein (HDL).(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance studies of mast cell histamine

The state of histamine in mast cells was studied by 1H NMR spectroscopy. Spectra were measured for histamine in situ in intact mast cells, for histamine in suspensions of mast cell granule matrices that had been stripped of their membranes, and for histamine in solutions of heparin. The 1H NMR spectrum of intact mast cells is relatively simple, consisting predominantly of resonances for intracellular histamine superimposed on a weaker background of resonances from heparin and proteins of the cells. All of the intracellular histamine contributes to the NMR signals, indicating it must be relatively mobile and not rigidly associated with the negatively charged granule matrix. Spectra for intracellular histamine and for histamine in granule matrices are similar, indicating the latter to be a reasonable model for the in situ situation. The dynamics of binding of histamine by granule matrices and by heparin are considerably different; exchange of histamine between the bulk water and the granule matrices is slow on the 1H NMR time scale, whereas exchange between the free and bound forms in heparin solution is fast. The chemical shifts of resonances for histamine in mast cells are pH dependent, decreasing as the intragranule pH increases without splitting or broadening. The results are interpreted to indicate that histamine in mast cells is relatively labile, with rapid exchange between bound histamine and pools of free histamine in water compartments confined in the granule matrix.     

Lipid molecular motion and enzyme activity in sarcoplasmic reticulum membrane.

In biochemically active sarcoplasmic reticulum vesicles (SR) the physical state of the membrane lipids was studied by high angle x-ray diffraction and proton nuclear magnetic resonance (NMR) at 220 MHz, and related to thermal effects observed in SR functional parameters. It is shown by high angle x-ray diffraction that even at temperatures as low as 1 degree C nearly all the SR lipid hydrocarbon chains are in a disordered conformation and only a very small part (less than 3%) are in rigid crystalline order. Consistent with this observation, the NMR data indicate that the majority of SR phospholipid molecules are in a state of restricted anisotropic motion having no apparent crystalline order at temperatures as low as 5 degrees C. At this temperature most of the resonance signal is contained in a broad feature-less line of 700-Hz half-width. On the other hand, as the temperature is raised, high-resolution NMR signals, representing groups with highly isotropic motion, begin to grow in intensity. It is estimated that by 35 degrees C 90-100% of the phosphatidylcholine N-methyl protons and 35% of the hydrocarbon-chain protons give high-resolution signals. Concurrent studies on functional parameters reveal thermal effects giving rise to nonlinear Arrhenius plots for the rates of calcium transport and calcium activated ATPase. The thermal effects observed on functional parameters and on the character of phospholipid molecular motion exhibit a parallel behavior, suggesting a relationship between enzyme activity and the physical state of the membrane lipids.     

The lipid composition of isolated cytoplasmic lipid droplets from a human cancer cell line, BE(2)M17

(1)H nuclear magnetic resonance spectroscopy (NMR) resonances from lipids in tumours are associated with tumour grade and treatment response. The origin of these NMR signals is mainly considered to be cytoplasmic lipid droplets (LDs). Techniques exist for isolating LDs but little is known about their composition and its relationship to NMR signals. In this work, density-gradient ultracentrifugation was performed on homogenised human cancer cells to isolate LDs. (1)H NMR was performed on whole cells, isolated LDs and their extracts. Heteronuclear single quantum coherence spectroscopy (HSQC) and liquid chromatography mass spectroscopy (LC-MS) were performed on lipid extracts of LDs. Staining and microscopy were used to characterize isolated LDs. An excellent agreement in chemical shift and relative signal intensity was observed between lipid resonances in cells and isolated LD spectra supporting that NMR-visible lipids originate primarily from LDs. Isolated LDs showed high concentrations of unsaturated lipids, a oleic-to-linoleic acid ratio greater than two and a cholesteryl ester (ChE)-to-cholesterol (Ch) ratio close to unity. These ratios were several-fold greater than respective ratios in whole cells, demonstrating isolation is important to characterize LD composition. LDs contain a specific group of lipid species that are likely to contribute to the (1)H NMR spectrum of cells.     

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity ¹. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations ^1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted ³. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals ⁴. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm^-1 CH stretch vibration in lipids and the 2120 cm^-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule''s vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.     

Studies of interactions of carbonic anhydrase B with water and urea: II. Spin diffusion of associates of protein intermediate state at 4.2 M of urea

The formation of carbonic anhydrase B associates (pH 5.7, urea concentration 4.2 M, 297 K) was studied as a function of protein concentration and time by nuclear magnetic resonance spectroscopy (spin diffusion method). It was found that the association process proceeds in two steps. The first step is relatively fast and cannot be controlled by our methods. During this step, persistent units are built. These consist of protein molecules that are able to interact with solvent molecules and with each other when protein solution contains 4.2 M of urea. Persistent units are relatively small (two, three protein molecules), and their mobility matches one of a single protein. The second step is slower, and throughout this step large structures are formed from persistent units. The parameters G* and S*, which characterize spin diffusion in a protein and a solvent, respectively (when spin diffusion excitation happens away from NMR spectral signals) are related to the probable size distribution of protein-solvent associates and are determined by their collective properties.     

The depletion of protein signals in metabonomics analysis with the WET-CPMG pulse sequence

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool capable of providing a comprehensive metabolic profile of biofluids such as urine, plasma, and serum. Unfortunately, when measuring serum and plasma, the high protein concentration can obscure the signals originating from low molecular weight metabolites. We evaluated the use of different parameters within the Carr-Purcell-Meiboom-Gill (CPMG) pulse train of fast spin-echoes to remove the macromolecular signal contribution in one-dimensional proton (1H) NMR spectra. Experimental parameters such as the refocusing delay in the CPMG pulse train, pulse miscalibration, and recycle time were examined to assess the ability to remove the protein signals from the spectrum without causing a deleterious effect on the signals originating from free, low molecular weight metabolites. The 1H-NMR spectra of a variety of serum samples spiked with 2'-deoxyadenosine were acquired using various acquisition parameters. Our results show that the delay used in the CPMG spin-echo and the combination of the acquisition pulse flip angle and recycle time are the two major factors affecting the observed metabolite signal amplitudes in the resulting 1H-NMR spectrum.     

Signaling and transport processes related to the carnivorous lifestyle of plants living on nutrient-poor soil

In Eukaryotes, long-distance and rapid signal transmission is required in order to be able to react fast and flexibly to external stimuli. This long-distance signal transmission cannot take place by diffusion of signal molecules from the site of perception to the target tissue, as their speed is insufficient. Therefore, for adequate stimulus transmission, plants as well as animals make use of electrical signal transmission, as this can quickly cover long distances. This update summarises the most important advances in plant electrical signal transduction with a focus on the carnivorous Venus flytrap. It highlights the different types of electrical signals, examines their underlying ion fluxes and summarises the carnivorous processes downstream of the electrical signals.     

NMR characterization of the pH 4 beta-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility

Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely alpha-helical isoform to a beta-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an alpha-helical protein into one that contains a high proportion of beta-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. (15)N-HSQC (heteronuclear single-quantum correlation) studies with mPrP (mouse PrP)-(23-231) show that a total of 150 dispersed amide signals are resolved in the native form, whereas only 65 amide signals with little chemical shift dispersion are observable in the pH 4 form. Three-dimensional (15)N-HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus: residues 23-118. (15)N transverse relaxation measurements indicate that these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP-(112-231), which shows only 16 (15)N-HSQC amide peaks at pH 4. The loss of signals from the C-terminus can be attributed to line broadening due to an increase in the molecular size of the oligomer or exchange broadening in a molten-globule state.     

An NMR method for studying the kinetics of metal exchange in biomolecular systems

The kinetics of lanthanide (III) exchange for calcium(II) in the C-terminal EF-hand of the protein calbindin D_9khave been studied by one-dimensional (1D) stopped-flow NMR. By choosing a paramagnetic lanthanide (Ce³⁺), kinetics in the sub-second range can be easily measured. This is made possible by the fact that (i) the kinetic behaviour of hyperfine shifted signals can be monitored in 1D NMR and (ii) fast repetition rates can be employed because these hyperfine shifted signals relax fast. It is found that the Ce³⁺-Ca²⁺exchange process indeed takes place on a sub-second timescale and can be easily monitored with this technique. As the rate of calcium-cerium substitution was found not to depend on the presence of excess calcium in solution, the kinetics of the process were interpreted in terms of a bimolecular associative mechanism, and the rate constants extracted. Interestingly, the dissociative mechanism involving the apoform of the protein, which is generally assumed for metal ion exchange at protein binding sites, was not in agreement with our data.     

The ER-mitochondria interface: the social network of cell death

When cellular organelles communicate bad things can happen. Recent findings uncovered that the junction between the endoplasmic reticulum (ER) and the mitochondria holds a crucial role for cell death regulation. Not only does this locale connect the two best-known organelles in apoptosis, numerous regulators of cell death are concentrated at this spot, providing a terrain for intense signal transfers. Ca2+ is the most prominent signalling factor that is released from the ER and, at high concentration, mediates the transfer of an apoptosis signal to mitochondria as the executioner organelle for cell death. An elaborate array of checks and balances is fine-tuning this process including Bcl-2 family members. Moreover, MAMs, "mitochondria-associated membranes", are distinct membrane sections at the ER that are in close contact with mitochondria and have been found to exchange lipids and lipid-derived molecules such as ceramide for apoptosis induction. Recent work has also described a reverse transfer of apoptosis signals, from mitochondria to the ER, via cytochrome c release and prolonged IP3R opening or through the mitochondrial fission factor Fis1 and Bap31 at the ER, which form the ARCosome, a novel caspase-activation complex.     

Detection of site-specific binding and co-binding of ligands to macromolecules using 19F NMR

Study of ligand-macromolecular interactions by 19F nuclear magnetic resonance (NMR) spectroscopy affords many opportunities for obtaining molecular biochemical and pharmaceutical information. This is due to the absence of a background fluorine signal, as well as the relatively high sensitivity of 19F NMR. Use of fluorine-labeled ligands enables one to probe not only binding and co-binding phenomena to macromolecules, but also can provide data on binding constants, stoichiometries, kinetics, and conformational properties of these complexes. Under conditions of slow exchange and macromolecule-induced chemical shifts, multiple 19F NMR resonances can be observed for free and bound ligands. These shifted resonances are a direct correlate of the concentration of ligand bound in a specific state rather than the global concentrations of bound or free ligand which are usually determined using other techniques such as absorption spectroscopy or equilibrium dialysis. Examples of these interactions are demonstrated both from the literature and from interactions of 5-fluorotryptophan, 5-fluorosalicylic acid, flurbiprofen, and sulindac sulfide with human serum albumin. Other applications of 19F NMR to study of these interactions in vivo, as well for receptor binding and metabolic tracing of fluorinated drugs and proteins are discussed.     

Programming xenon diffusion in maltose-binding protein

Protein interiors contain void space that can bind small gas molecules. Determination of gas pathways and kinetics in proteins has been an intriguing and challenging task. Here, we combined computational methods and the hyperpolarized xenon-129 chemical exchange saturation transfer (hyper-CEST) NMR technique to investigate xenon (Xe) exchange kinetics in maltose-binding protein (MBP). A salt bridge ～9 Å from the Xe-binding site formed upon maltose binding and slowed the Xe exchange rate, leading to a hyper-CEST ¹²⁹Xe signal from maltose-bound MBP. Xe dissociation occurred faster than dissociation of the salt bridge, as shown by ¹³C NMR spectroscopy and variable-B₁ hyper-CEST experiments. “Xe flooding” molecular dynamics simulations identified a surface hydrophobic site, V23, that has good Xe binding affinity. Mutations at this site confirmed its role as a secondary exchange pathway in modulating Xe diffusion. This shows the possibility for site-specifically controlling xenon protein-solvent exchange. Analysis of the available MBP structures suggests a biological role of MBP’s large hydrophobic cavity to accommodate structural changes associated with ligand binding and protein-protein interactions.     

Time-resolved NMR studies of RNA folding

The application of real-time NMR experiments to the study of RNA folding, as reviewed in this article, is relatively new. For many RNA folding events, current investigations suggest that the time scales are in the second to minute regime. In addition, the initial investigations suggest that different folding rates are observed for one structural transition may be due to the hierarchical folding units of RNA. Many of the experiments developed in the field of NMR of protein folding cannot directly be transferred to RNA: hydrogen exchange experiments outside the spectrometer cannot be applied since the intrinsic exchange rates are too fast in RNA, relaxation dispersion experiments on the other require faster structural transitions than those observed in RNA. On the other hand, information derived from time-resolved NMR experiments, namely the acquisition of native chemical shifts, can be readily interpreted in light of formation of a single long-range hydrogen bonding interaction. Together with mutational data that can readily be obtained for RNA and new ligation technologies that enhance site resolution even further, time-resolved NMR may become a powerful tool to decipher RNA folding. Such understanding will be of importance to understand the functions of coding and non-coding RNAs in cells.     

Application of osmometric methods to the study of lipids, lipoproteins, and apolipoproteins

Membrane and vapor pressure osmometry are two colligative methods that can be useful in lipid research. The former method can be used to study proteins or other macromolecules whose molecular weight lies between 20,000 to 1,000,000. Vapor pressure osmometry is useful with smaller molecules having a molecular weight of 10,000 or less. These techniques can be used in aqueous or nonaqueous solutions. They are rapid, precise, nondestructive, and require relatively small amounts of material. These techniques provide information about the state of aggregation and also about interactions of lipids, lipoproteins, and apolipoproteins in solution. We will show how membrane osmometry can be used to study solutions of lipoproteins and apolipoproteins. The application of vapor pressure osmometry to the study of biologically important lipids such as cholesterol, cholesteryl esters, and bile salts is shown.     

A minisensor for the rapid screening of sucralose based on surface-stabilized bilayer lipid membranes.

This work describes an electrochemical technique that is suitable for the rapid and sensitive screening of the sweetener sucralose based on surface-stabilized bilayer lipid membranes (s-BLMs) composed of egg phosphatidylcholine. The interactions of sucralose with s-BLMs produced electrochemical ion current increases, which appeared reproducible within a few seconds after exposure of the membranes to the sweetener. The mechanism of signal generation was investigated by differential scanning calorimetric studies. The mechanism was found to be associated with alteration of the electrostatic fields of the lipid film. These studies revealed that an increase of the molecular area of the lipids at the membranes and a stabilization of a gel phase structure occurred due to adsorption of the sweetener. Water molecules are adsorbed at the polar headgroups of the lipids, which changes the electrostatic field at the surface of the membranes. The current signal increases were related to the concentration of sucralose in bulk solution in the micromolar range. The present lipid film based sensor provided a fast response (i.e. in the order of a few seconds) to alterations of sucralose concentration (5-50 microm) in electrolyte solution. The electrochemical transduction of the interactions of this artificial sweetener with s-BLMs was applied in the determination of this compound in granulated sugar substitute products using the present minisensor.     

Interaction of mitotic oscillators as a source of variability of cell cycle duration

Interaction between membrane mitotic oscillators at the expense of exchange with the molecules of lipids (slow variable) and antioxidants (fast variable) was considered. Parameters of all the oscillators are equal, excluding a small noise added to the equation for lipids. These parameters are chosen in such a way that the oscillators are not far from the transition to the stable stationary state. The numerical modeling has shown that the exchange with lipids brings about the appearance of an additional limit cycle whose period is significantly greater than that of an autonomous oscillator. The addition of noise averages the behaviour of oscillators, and distribution according to cycle duration becomes broad and bimodal. Thus the exchange of the slow variable increases the dispersion of distribution of cell generation times. This conclusion seems to be true for any oscillator with similar dynamic properties.     

Characterization of Stratum Corneum Molecular Dynamics by Natural-Abundance 13C Solid-State NMR

Despite the enormous potential for pharmaceutical applications, there is still a lack of understanding of the molecular details that can contribute to increased permeability of the stratum corneum (SC). To investigate the influence of hydration and heating on the SC, we record the natural-abundance ¹³C signal of SC using polarization transfer solid-state NMR methods. Resonance lines from all major SC components are assigned. Comparison of the signal intensities obtained with the INEPT and CP pulse sequences gives information on the molecular dynamics of SC components. The majority of the lipids are rigid at 32°C, and those lipids co-exist with a small pool of mobile lipids. The ratio between mobile and rigid lipids increases with hydration. An abrupt change of keratin filament dynamics occurs at RH = 80–85%, from completely rigid to a structure with rigid backbone and mobile protruding terminals. Heating has a strong effect on the lipid mobility, but only a weak influence on the keratin filaments. The results provide novel molecular insight into how the SC constituents are affected by hydration and heating, and improve the understanding of enhanced SC permeability, which is associated with elevated temperatures and SC hydration.