Molecular dynamics simulation of single-walled carbon nanotubes inside liquid crystals

Molecular dynamic simulations of systems of single-walled carbon nanotubes (CNTs) in liquid crystalline solvents were performed, in order to investigate the effect of the molecular structure and phase of the liquid crystal (LC) on the interactions between the CNTs. Three different LC molecules (5CB, 8CB and 5CF) were considered in our study. Our results with 5CB and 8CB suggest that increasing the chain length of the hydrophobic part of the LC molecule by three carbon atoms is insufficient to decrease the tendency for the CNTs to aggregate in the LCs. Additionally, varying the phase of the LC is also insufficient to decrease the aggregation tendency of the CNTs. However, simulations with 5CF (which has fluorine atoms in the head group of the LC molecule) suggest that this LC solvent can decrease the tendency of the CNTs to aggregate. This study is relevant to assist experimentalists with the development of high-quality dispersions of large concentrations of CNTs in the LCs.     

Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values

It is demonstrated that mixtures of ditetradecyl- phosphatidylcholine or didodecyl-phoshatidylcholine and dihexyl- phosphatidylcholine in water form lyotropic liquid crystalline phases under similar conditions as previously reported for bicelles consisting of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl- phosphatidylcholine (DHPC). The carboxy-ester bonds present in DMPC and DHPC are replaced by ether linkages in their alkyl analogs, which prevents acid- or base-catalyzed hydrolysis of these compounds. 15N-1H dipolar couplings measured for ubiquitin over the 2.3–10.4pH range indicate that this protein retains a backbone conformation which is very similar to its structure at pH 6.5 over this entire range.     

Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules

Weak alignment of solute molecules with the magnetic field can be achieved in a dilute liquid crystalline medium, consisting of an aqueous mixture of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). For a certain range of molar ratios, DMPC and DHPC can form large, disc-shaped particles, commonly referred to as bicelles (Sanders and Schwonek, 1992), which cooperatively align in the magnetic field and induce a small degree of alignment on asymmetrically shaped solute molecules. As a result, dipolar couplings between pairs of ¹H, ¹³C or ¹⁵N nuclei are no longer averaged to zero by rotational diffusion and they can be readily measured, providing valuable structural information. The stability of these liquid crystals and the degree of alignment of the solute molecules depend strongly on experimental variables such as the DMPC:DHPC ratio and concentration, the preparation protocol of the DMPC/DHPC mixtures, as well as salt, temperature, and pH. The lower temperature limit for which the liquid crystalline phase is stable can be reduced to 20 °C by using a ternary mixture of DHPC, DMPC, and 1-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine, or a binary mixture of DHPC and ditridecanoyl-phosphatidylcholine. These issues are discussed, with an emphasis on the use of the medium for obtaining weak alignment of biological macromolecules.     

Structure and dynamics of melittin in lysomyristoyl phosphatidylcholine micelles determined by nuclear magnetic resonance.

Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25 degrees C were investigated by (13)C- and (31)P-NMR spectroscopy. (13)C alpha chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly alpha-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. (31)P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. (13)C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S(2)) of C alpha-H vectors averaged 0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide C alpha moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S(2) values for the C epsilon-H vectors being 0.19, 0.30, and 0.44 for lys-7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion tau e were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S(2) for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lysine side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.     

A simple apparatus for generating stretched polyacrylamide gels, yielding uniform alignment of proteins and detergent micelles*

Compressed and stretched polyacrylamide hydrogels previously have been shown to offer a robust method for aligning proteins. A simple, funnel-like apparatus is described for generating uniformly stretched hydrogels. For prolate-shaped proteins, gels stretched in the direction of the magnetic field yield two-fold larger alignment than gels compressed to the same aspect ratio in this direction. Empirically, protein alignment is found to be proportional to (c–2.3)² [(d_o/d_N)³-1], where d_o and d_N are the diameters of the cylindrical gels before and after stretching, respectively, and c is the polyacrylamide weight fraction in percent. Low gel densities, in the 4–7% range, are found to have minimal effects on macromolecular rotational correlation times, _c, and no effect of the compression ratio on _c could be discerned over the range studied (d_o/d_N le1.4). Application is demonstrated for a sample containing the first Ig-binding domain of protein G, and for a detergent-solubilized peptide.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

Use of fully deuterated micelles for conformational studies of membrane proteins by high resolution 1H nuclear magnetic resonance

Micellar complexes of melittin with fully deuterated detergents have been studied by high resolution ¹H nuclear magnetic resonance (NMR). The synthesis of deuterated micelles is described and it is shown that the ¹H NMR spectrum of micelle-bound melittin is well resolved and suitable for detailed analysis by conventional high-resolution NMR methods. A preliminary characterization of micelle-bound melittin shows that interaction with the micelle results in different conformational and dynamic features for the hydrophobic and hydrophilic regions of the melittin amino acid sequence. The present experiments on melittin and preliminary results with other polypeptides and proteins demonstrate that in favourable cases high-resolution ¹H NMR studies of the complexes formed between membrane proteins and deuterated micelles provides a viable method for conformational studies of membrane-bound proteins.     

Technological advancements in bio-recognition using liquid crystals: Techniques,applications, and performance

The application of liquid crystal (LC) materials has undergone a modern-day renaissance from its classical use in electronics industry as display devices to new-fangled techniques for optically detecting biological and chemical analytes. This review article deals with the emergence of LC materials as invaluable material for their use as label-free sensing elements in the development of optical, electro-optical and electrochemical biosensors. The property of LC molecules to change their orientation on perturbation by any external stimuli or on interaction with bioanalytes or chemical species has been utilized by many researches for the fabrication of high sensitive LC-biosensors. In this review article we categorized LC-biosensor based on biomolecular reaction mechanism viz. enzymatic, nucleotides and immunoreaction in conjunction with operating principle at different LC interface namely LC-solid, LC-aqueous and LC-droplets. Based on bimolecular reaction mechanism, the application of LC has been delineated with recent progress made in designing of LC-interface for the detection of bio and chemical analytes of proteins, virus, bacteria, clinically relevant compounds, heavy metal ions and environmental pollutants. The review briefly describes the experimental set-ups, sensitivity, specificity, limit of detection and linear range of various viable and conspicuous LC-based biosensor platforms with associated advantages and disadvantages therein.     

Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone ¹⁵N T ₁/T ₂ data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T ₁/T ₂ ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.     

Dilute liquid crystals used to enhance residual dipolar couplings may alter conformational equilibrium in oligosaccharides

The solution structures of a trisaccharide and a pentasaccharide containing the Lewis(x) motif were determined by two independent approaches using either dipolar cross-relaxation (NOE) or residual dipolar coupling (RDC) data. For the latter, one-bond 13C[bond](1)H RDC enhanced by two different mineral liquid crystals were used alone. Home-written programs were employed firstly for measuring accurately the coupling constants in the direct dimension of non-decoupled HSQC experiments, secondly for transforming each RDC data set into geometrical restraints. In this second program, the complete molecular structure was expressed in a unique frame where the alignment tensor is diagonal. Assuming that the pyranose rings are rigid, their relative orientation is defined by optimizing the glycosidic torsion angles. For the trisaccharide, a good agreement was observed between the results of both approaches (NOE and RDC). In contrast, for the pentasaccharide, strong discrepancies appeared, which seem to result from interactions between the pentasaccharide and the mesogens, affecting conformational equilibrium. This observation is of importance, as it reveals that using simultaneously NOE and RDC can be hazardous as the former represent 99% of the molecules free in solution, whereas the latter correspond to less than 1% of the structure bound to the mesogen.     

Characterization of surfactant liquid crystal phases suitable for molecular alignment and measurement of dipolar couplings

Media employed for imparting partial alignment onto solute molecules have recently attracted considerable attention, since they permit the measurement of NMR parameters for solute biomolecules commonly associated with solid state NMR. Here we characterize a medium which is based on a quasi-ternary surfactant system comprising cetylpyridinium bromide/hexanol/sodium bromide. We demonstrate that dilute solutions of this system can exist in liquid crystalline phases which orient in the magnetic field and allow the measurement of residual dipolar couplings under a variety of conditions. The present system is extremely versatile and robust, tolerating different buffer conditions, temperature ranges and concentrations.     

Nuclear magnetic relaxation dispersion study of the dynamics in solid homopolypeptides

The (1)H nuclear magnetic relaxation dispersion profiles were measured from 10 kHz to 30 MHz as a function of temperature for polyglycine, polyalanine, polyvaline, and polyphenylalanine to examine the contributions of different side chain motions to the polypeptide proton relaxation rate constants. The spin-fracton theory for (1)H relaxation is modified to account for high frequency motions of side chains that are dynamically connected to the linear polymer backbone. The (1)H relaxation is dominated by propagation of rare disturbances along the backbone of the polymer. The side-chain dynamics cause an off-set in the field dependence of the (1)H spin-lattice relaxation rate constants which obey a power law in the Larmor frequency in the limit of low and high magnetic field strength.     

Overall structure and sugar dynamics of a DNA dodecamer from homo- and heteronuclear dipolar couplings and 31P chemical shift anisotropy

The solution structure of d(CGCGAATTCGCG)₂ has been determined on the basis of an exceptionally large set of residual dipolar couplings. In addition to the heteronuclear ¹³C-¹H and ¹⁵N-¹H and qualitative homonuclear ¹H-¹H dipolar couplings, previously measured in bicelle medium, more than 300 quantitative ¹H-¹H and 22 ³¹P-¹H dipolar restraints were obtained in liquid crystalline Pf1 medium, and 22 ³¹P chemical shift anisotropy restraints. High quality DNA structures can be obtained solely on the basis of these new restraints, and these structures are in close agreement with those calculated previously on the basis of ¹³C-¹H and ¹⁵N-¹H dipolar couplings. In the newly calculated structures, ³¹P-¹H dipolar and ³Jsub H3 P sub couplings and ³¹P CSA data restrain the phosphodiester backbone torsion angles. The final structure represents a quite regular B-form helix with a modest bending of 10°, which is essentially independent of whether or not electrostatic terms are used in the calculation. Combined, the number of homo- and heteronuclear dipolar couplings significantly exceeds the number of degrees of freedom in the system. Results indicate that the dipolar coupling data cannot be fit by a single structure, but are compatible with the presence of rapid equilibria between C2-endo and C3-endo deoxyribose puckers (sugar switching). The C2-H2/H2 dipolar couplings in B-form DNA are particularly sensitive to sugar pucker and yield the largest discrepancies when fit to a single structure. To resolve these discrepancies, we suggest a simplified dipolar coupling analysis that yields N/S equilibria for the ribose sugar puckers, which are in good agreement with previous analyses of NMR J_HH couplings, with a population of the minor C3-endo form higher for pyrimidines than for purines.     

Structure and dynamics of a membrane protein in micelles from three solution NMR experiments

Three solution NMR experiments on a uniformly ¹⁵N labeled membrane protein in micelles provide sufficient information to describe the structure, topology, and dynamics of its helices, as well as additional information that characterizes the principal features of residues in terminal and inter-helical loop regions. The backbone amide resonances are assigned with an HMQC-NOESY experiment and the backbone dynamics are characterized by a ¹H-¹⁵N heteronuclear NOE experiment, which clearly distinguishes between the structured helical residues and the more mobile residues in the terminal and interhelical loop regions of the protein. The structure and topology of the helices are described by Dipolar waves and PISA wheels derived from experimental measurements of residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). The results show that the membrane-bound form of Pf1 coat protein has a 20-residue trans-membrane hydrophobic helix with an orientation that differs by about 90° from that of an 8-residue amphipathic helix. This combination of three-experiments that yields Dipolar waves and PISA wheels has the potential to contribute to high-throughput structural characterizations of membrane proteins.     

Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR

Understanding protein stability requires characterization of structural determinants of the folded and unfolded states. Many proteins are capable of populating partially folded states under specific solution conditions. Occasionally, coexistence of the folded and an unfolded state under non- or mildly denaturing conditions can be observed by NMR, allowing us to structurally probe these states under identical conditions. Here we report on a destabilized mutant of the B1 domain of protein G (GB1) whose equilibrium unfolding was systematically investigated. Backbone amide residual dipolar couplings (RDCs), the tryptophan Nepsilon-H resonance and the amide nitrogen transverse relaxation rates (R2s) for varying pH values and different temperatures were measured. The backbone amide RDCs indicate that prior to complete unfolding, two melting hot spots are formed at the turn around T11, L12 and K13 and the N terminus of the helix at A24 and T25. The RDCs for the low pH, thermally unfolded state of GB1 are very small and do not indicate the presence of any native-like structure. Amide nitrogen transverse relaxation rates for GB1 in the folded state at different temperatures exhibit large contributions from exchange processes and the associated dynamics display considerable heterogeneity. Our data provide clear evidence for intermediate conformations and multi-state equilibrium un/folding for this GB1 variant.     

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.     

Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the ²H NMR water signal and by the measurement of ¹H-¹⁵N residual dipolar couplings (RDC) in the archeal translation elongation factor 1. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample.     

蛋白质溶液NMR结构测定的一些新进展

新的标记技术的进展和采用稀释的液晶作为溶剂以提供额外的结构信息,提高了核磁共振技术测定蛋白质溶液三维结构的精度,扩大了分子质量测定范围.目前已经利用多维 ¹⁵N,¹³C,²H标记NMR测定了许多分子质量为30 ku左右的蛋白质溶液结构,这一上限可能还会被进一步提高.     

Study of conformational rearrangement and refinement of structural homology models by the use of heteronuclear dipolar couplings

For an increasing fraction of proteins whose structures are being studied, sequence homology to known structures permits building of low resolution structural models. It is demonstrated that dipolar couplings, measured in a liquid crystalline medium, not only can validate such structural models, but also refine them. Here, experimental ¹H-¹⁵N, ¹H-¹³C, and ¹³C-¹³C dipolar couplings are shown to decrease the backbone rmsd between various homology models of calmodulin (CaM) and its crystal structure. Starting from a model of the Ca²⁺-saturated C-terminal domain of CaM, built from the structure of Ca²⁺-free recoverin on the basis of remote sequence homology, dipolar couplings are used to decrease the rmsd between the model and the crystal structure from 5.0 to 1.25 Å. A better starting model, built from the crystal structure of Ca²⁺-saturated parvalbumin, decreases in rmsd from 1.25 to 0.93 Å. Similarly, starting from the structure of the Ca²⁺-ligated CaM N-terminal domain, experimental dipolar couplings measured for the Ca²⁺-free form decrease the backbone rmsd relative to the refined solution structure of apo-CaM from 4.2 to 1.0 Å.