Orientation Of Helices Research Articles

Influence of interfacial tryptophan residues on an arginine-flanked transmembrane helix

The transmembrane helices of membrane proteins often are flanked by interfacial charged or aromatic residues that potentially help to anchor the membrane-spanning protein. For isolated single-span helices, the interfacial residues may be especially important for stabilizing particular tilted transmembrane orientations. The peptide RWALP23 (acetyl-GR2ALW(LA)6LWLAR22A-amide) has been employed to investigate the interplay between interfacial arginines and tryptophans. Here we replace the tryptophans of RWALP23 with A5 and A19, to investigate arginines alone with respect to helix fraying and orientation in varying lipid bilayers. Deuterated alanines incorporated into the central sequence allow the orientation and stability of the core helix to be assessed by means of solid -state 2H NMR in bilayers of DOPC, DMPC and DLPC. The helix tilt from the bilayer normal is found to increase slightly when R2 and R22 are present, and increases still further when the tryptophans W5 and W19 are replaced by alanines. The extent of helix dynamic averaging remains low in all cases. The preferred helix azimuthal rotation is essentially constant for all of the helices in each of the lipid membranes considered here. The alanines located outside of the core region of the peptide are sensitive to helical integrity. The new alanines, A5 and A19, therefore, provide new information about the length of the core helix and the onset of unraveling of the terminals. Residue A19 remains essentially on the central helix in each lipid membrane, while residues A3, A5 and A21 deviate from the core helix to an extent that depends on the membrane thickness. Differential unraveling of the two ends to expose peptide backbone groups for hydrogen bonding therefore acts together with specific interfacial side chains to stabilize a transmembrane helix.

Role of membrane curvature on the activation/deactivation of Carnitine Palmitoyltransferase 1A: A coarse grain molecular dynamic study

Carnitine Palmitoyltransferase 1A (CPT 1A) is an enzyme anchored to the outer mitochondrial membrane (OMM), where it regulates the passage of fatty acids into the mitochondria and intervenes in the process of β-oxidation of long-chain fatty acids. Although CPT 1A is inhibited by malonyl-CoA, its activity is also modulated by the curvature of OMM. This modulation depends on the behavior of the N-terminal domain (NTD), which can be adsorbed onto the OMM (nonactive CPT 1A) or interacting with the C-terminal domain (active CPT 1A). Aimed to provide mechanistic insights on the regulatory mechanism of CPT 1A, we studied the influence of the bilayer curvature on the NTD behavior through a series of coarse-grained (CG) molecular dynamics simulations using curved and planar membranes. Comparative analysis suggests that the main determinant for the activation/deactivation of the enzyme is the tilt angle orientation of the transmembrane (TM) domains. Planar membranes induce a wide variation on the tilt angle orientation of TM helices, while curved geometries promote small angles with the membrane normal. Our results identify the first TM domain as an important component of the membrane sensing mechanism.

Influence of Lipid Saturation, Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes.

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW5 LAL8 LALALAL16 ALW19 LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific 2 H-labeled alanine residues within the central sequence for detection by solid-state 2 H NMR spectroscopy. The resulting pattern of [2 H]Ala quadrupolar splitting (Δνq ) magnitudes indicates the core helix for R8,16 GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R8,16 GWALP23 helix adopts primarily a surface orientation. The inclusion of 10-20 mol % cholesterol in DOPC bilayers drives more of the R8,16 GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.

Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) to Determine the Topology Map and Probe the Solvent Accessibility of Membrane Proteins

The topology of helix-bundle membrane proteins provides low-resolution structural information with regard to the number and orientation of membrane-spanning helices, as well as the sidedness of intra/extra-cellular domains. In the past decades, several strategies have been developed to experimentally determine the topology of membrane proteins. However, generally, these methods are labour-intensive, time-consuming and difficult to implement for quantitative analysis. Here, we report a novel approach, site-directed alkylation detected by in-gel fluorescence (SDAF), which monitors the fluorescent band shift caused by alkylation of the EGFP-fused target membrane protein bearing one single introduced cysteine. In-gel fluorescence provides a unique readout of target membrane proteins with EGFP fusion from non-purified samples, revealing a distinct 5 kDa shift on SDS-PAGE gel due to conjugation with mPEG-MAL-5K. Using the structurally characterised bile acid transporter ASBTNM as an example, we demonstrate that SDAF generates a topology map consistent with the crystal structure. The efficiency of mPEG-MAL-5K modification at each introduced cysteine can easily be quantified and analysed, providing a useful tool for probing the solvent accessibility at a specific position of the target membrane protein.

Breaking the Backbone: Central Arginine Residues Induce Membrane Exit and Helix Distortions within a Dynamic Membrane Peptide.

Transmembrane domains of membrane proteins sometimes contain conserved charged or ionizable residues which may be essential for protein function and regulation. This work examines the molecular interactions of single Arg residues within a highly dynamic transmembrane peptide helix. To this end, we have modified the GW4,20ALP23 (acetyl-GGAW4(AL)7AW20AGA-amide) model peptide framework to incorporate Arg residues near the center of the peptide. Peptide helix formation, orientation and dynamics were analyzed by means of solid-state NMR spectroscopy to monitor specific 2H- or 15N-labeled residues. GW4,20ALP23 itself adopts a tilted orientation within lipid bilayer membranes. Nevertheless, the GW4,20ALP23 helix exhibits moderate to high dynamic averaging of NMR observables, such as 2H quadrupolar splittings or 15N-1H dipolar couplings, due to competition between the interfacial Trp residues on opposing helix faces. Here we examine how the helix dynamics are impacted by the introduction of a single Arg residue at position 12 or 14. Residue R14 restricts the helix to low dynamic averaging and a well-defined tilt that varies inversely with the lipid bilayer thickness. To compensate for the dominance of R14, the competing Trp residues cause partial unwinding of the helix at the C-terminal. By contrast, R12GW4,20ALP23 exits the DOPC bilayer to an interfacial surface-bound location. Interestingly, multiple orientations are exhibited by a single residue, Ala-9. Quadrupolar splittings generated by 2H-labeled residues A3, A5, A7, and A9 do not fit to the α-helical quadrupolar wave plot defined by residues A11, A13, A15, A17, A19, and A21. The discontinuity at residue A9 implicates a helical swivel distortion and an apparent 310-helix involving the N-terminal residues preceding A11. These molecular features suggest that, while arginine residues are prominent factors controlling transmembrane helix dynamics, the influence of interfacial tryptophan residues cannot be ignored.

Class Id ribonucleotide reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes on metal loading

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O2·−). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.

Methionine and Selenomethionine as Energy Transfer Acceptors for Biomolecular Structure Elucidation in the Gas Phase

Mass spectrometry affords rapid and sensitive analysis of peptides and proteins. Coupling spectroscopy with mass spectrometry allows for the development of new methods to enhance biomolecular structure determination. Herein, we demonstrate two new energy acceptors that can be utilized for action-excitation energy transfer experiments. In the first system, C-S bonds in methionine act as energy acceptors from native chromophores, including tyrosine, tryptophan, and phenylalanine. Comparison among chromophores reveals that tyrosine transfers energy most efficiently at 266nm, but phenylalanine and tryptophan also transfer energy with comparable efficiencies. Overall, the C-S bond dissociation yields following energy transfer are low for methionine, which led to an investigation of selenomethionine, a common analog that is found in many naturally occurring proteins. Sulfur and selenium are chemically similar, but C-Se bonds are weaker than C-S bonds and have lower lying σ* anti-bonding orbitals. Excitation of peptides containing tyrosine and tryptophan results in efficient energy transfer to selenomethionine and abundant C-Se bond dissociation. A series of helical peptides were examined where the positions of the donor or acceptor were systematically scanned to explore the influence of distance and helix orientation on energy transfer. The distance was found to be the primary factor affecting energy transfer efficiency, suggesting that selenomethionine may be a useful acceptor for probing protein structure in the gas phase.

Atomistic Models from Orientation and Distance Constraints Using EPR of a Bifunctional Spin Label

We have used high-resolution orientation and distance measurements derived from electron paramagnetic resonance of a bifunctional spin label (BSL) to build and refine atomistic models of protein structure. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin’s catalytic domain while myosin is in complex with actin. Constraints for orientation of individual helices were obtained in a previous study from continuous-wave electron paramagnetic resonance of myosin labeled at specific sites with BSLs in oriented muscle fibers. In this study, new distance constraints were derived from double electron-electron resonance on myosin constructs labeled with a BSL specifically at two sites. Using these complementary constraints together, we thoroughly characterize the BSL’s rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. We also leverage these measurements to derive a novel, to our knowledge, structural model for myosin-II in complex with actin and MgADP and compare our model to other recent actomyosin structures. The described approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di-Cys mutation is feasible.

Defect formation in N* LCs via photovoltaic fields: impact of surface treatment

ABSTRACTField-induced defect creation was investigated in chiral nematic liquid crystal test cells, where a Fe-doped lithium niobate thin-cut was applied as field generating substrate. These photo responsive test cells were exposed to focused continuous wave laser light, which resulted in the formation of localised defects. The covering indium tin oxide coated glass of the test cells was treated with a polyvinyl alcohol alignment layer. This alignment layer was either applied with a doctor blade technique or by spin casting of a polyvinyl solution, which was thermally annealed, subsequently. The surface roughness of the alignment layers was investigated with atomic force microscopy, which was interesting to understand how defects and locally lying helices were supported or suppressed in various samples. Well-confined circular defects were formed in test cells with spin-casted alignment layers and the appearance and unwanted interaction with oily streaks could be efficiently reduced in these samples. The results are promising. Samples with an oily streak-free homogenous standing helix orientation of the chiral nematic liquid crystal are an ideal canvas for rewritable pattern formation in a chiral nematic LC: Such samples have high potential as reconfigurable diffractive elements such as rewritable diffraction gratings.

Navigating the Structural Landscape of De Novo α-Helical Bundles.

The association of amphipathic α helices in water leads to α-helical-bundle protein structures. However, the driving force for this-the hydrophobic effect-is not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increasingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundle-the α-helical coiled coils-relationships have been established that discriminate between all-parallel dimers, trimers, and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less well understood. Here, we describe a synthetic-peptide system that switches between parallel hexamers and various up-down-up-down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high resolution with X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of α-helical coiled coils above the dimers and trimers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology.

High-Resolution Structural Studies Elucidate Antiatherogenic and Anti-Inflammatory Properties of Peptides Designed to Mimic Amphipathic α-Helical Domains of Apolipoprotein A-I.

Peptides designed to mimic the antiatherogenic and anti-inflammatory properties of apolipoprotein A-I show that although lipid association is required, not all lipid-associating peptides exhibit these properties. Our studies of a series of peptides showed that peptides with aromatic residues at the center of the nonpolar face were able to interact with inflammatory lipids and inhibited inflammation, which resulted in the amelioration of several lipid-mediated disorders such as lesion development, tumor formation, and Alzheimer’s plaque formation. The pKa values determined using 13C nuclear magnetic resonance (NMR) spectroscopy of K residues located at the polar-nonpolar interface provided the first clue to the relative orientations of the peptide helices with respect to each other and around the edge of the lipid discoidal complexes. High-resolution 1H-NMR studies of peptide-lipid discoidal complex confirmed the amphipathic α-helical structure of the peptide, location of aromatic residues of the peptide closer to the polar-nonpolar interface, and head-to-tail arrangement of the peptide helices around the edge of the disc. Amphipathic α-helical structure and the location of aromatic residues (F, W, Y) closer to the polar-nonpolar interface in a lipid environment allow the peptide to strongly bind oxidized lipids resulting in its anti-inflammatory properties.

Augmenting Bragg Reflection with Polymer-sustained Conical Helix

There has been a recent surge of interest in smart materials and devices with stimuli-responsive properties for optical modulations. Cholesteric liquid crystals (CLCs) are a unique class of light-manipulating materials, and strongly interact with light and other electromagnetic (EM) waves. Because of their intricate helical structure, new properties of CLC have emerged revealing unique optical behavior that has resulted in rewriting Braggs’ law for how light interacts with soft materials. The aim of this work is to push the limits of spectral tuning with a new method of augmenting light-cholesteric interactions using a polymer-sustained conical helix (PSCH) structure. We experimentally explore the reversibility of reflective wavelength modulation and validate the mechanism enhanced by a polymer-sustained helicoidal structure via theoretical analyses. The conical helix structure of a CLC, formed by low-field-induced oblique orientation of cholesteric helices, is comprised of a chiral dopant, a conventional nematic, and bimesogenic and trimesogenic nematics. Polymerizing a small amount of a reactive mesogen in the CLC with an applied electric field produces a templated helical polymer network that enables three switched optical states, including light-scattering and transparent states as well as color reflection in response to an applied increasing or decreasing electric field. An electro-activated PSCH optical film covers a wide color space, which is appropriate for tunable color device applications. We envisage that this PSCH material will lead to new avenues for controlling EM waves in imaging and thermal control, smart windows and electronic papers.

Tribo-functional effects of double-crossed helix on surface finish, cutting friction and tool wear mechanisms during the milling process of natural fiber composites

Tribological mechanisms that control the machining of natural fiber reinforced plastic (NFRP) composites are different from those of synthetic fiber composites in terms of cutting forces, tool wear, and surface finish. The aim of this paper is to investigate the cutting performances of NFRP composites using improved milling tool geometry with double-crossed helix (DCH). Cutting edges are coated with PVD-TiAlN thin coating thickness to ensure low cutting edge radius. Milling experiments are performed on unidirectional flax fibers reinforced polypropylene composites with CNC machine varying the tool kinematics. The in-situ cutting forces are acquired to evaluate the tribological performances of machining NFRP with DCH tool. The machined surfaces are rated by Scanning Electron Microscope (SEM) and peripheral surface defects are rated using an optical microscope. Moreover, the tool wear is evaluated at the cutting tool edge by SEM observations after accelerated wear tests. Results show that the improved milling tool overcomes the disadvantages and limitations of known NFRP machining in terms of surface finish. The functional design of the DCH generates a specific tool wear behavior where the wear mechanisms differ at each cutting helix orientation.

Helix Fraying and Orientation of a Transmembrane Peptide having a Long Hydrophobic Core and Anchored by Interfacial Arginine Residues

Helix Fraying and Orientation of a Transmembrane Peptide having a Long Hydrophobic Core and Anchored by Interfacial Arginine Residues

Conformations of an RNA Helix-Junction-Helix Construct Revealed by SAXS Refinement of MD Simulations

RNA is involved in a broad range of biological processes that extend far beyond translation. Many of RNA’s recently discovered functions rely on folding to a specific conformation or transitioning between conformations. The RNA structure contains rigid, short basepaired regions connected by more flexible linkers. Studies of model constructs such as small helix-junction-helix (HJH) motifs are useful in understanding how these elements work together to determine RNA conformation. Here, we reveal the full ensemble of solution structures assumed by a model RNA HJH. We apply small-angle x-ray scattering and an ensemble optimization method to selectively refine models generated by all-atom molecular dynamics simulations. The expectation of a broad distribution of helix orientations, at and above physiological ionic strength, is not met. Instead, this analysis shows that the HJH structures are dominated by two distinct conformations at moderate to high ionic strength. Atomic structures, selected from the molecular dynamics simulations, reveal strong base-base interactions in the junction that critically constrain the conformational space available to the HJH molecule and lead to a surprising re-extension at high salt. These results are corroborated by comparison with previous single-molecule fluorescence resonance energy transfer experiments on the same constructs.

How to fold and protect mitochondrial ribosomal RNA with fewer guanines.

Mammalian mitochondrial ribosomes evolved from bacterial ribosomes by reduction of ribosomal RNAs, increase of ribosomal protein content, and loss of guanine nucleotides. Guanine is the base most sensitive to oxidative damage. By systematically comparing high-quality, small ribosomal subunit RNA sequence alignments and solved 3D ribosome structures from mammalian mitochondria and bacteria, we deduce rules for folding a complex RNA with the remaining guanines shielded from solvent. Almost all conserved guanines in both bacterial and mammalian mitochondrial ribosomal RNA form guanine-specific, local or long-range, RNA–RNA or RNA–protein interactions. Many solvent-exposed guanines conserved in bacteria are replaced in mammalian mitochondria by bases less sensitive to oxidation. New guanines, conserved only in the mitochondrial alignment, are strategically positioned at solvent inaccessible sites to stabilize the ribosomal RNA structure. New mitochondrial proteins substitute for truncated RNA helices, maintain mutual spatial orientations of helices, compensate for lost RNA–RNA interactions, reduce solvent accessibility of bases, and replace guanines conserved in bacteria by forming specific amino acid–RNA interactions.

An Allosteric Glucagon Receptor Antagonist, LGD-6972, Displays Biased Receptor Signaling

Glucagon receptor antagonists (GRA) are novel molecules in development for the treatment of type 1 and type 2 (T2DM) diabetes. LGD-6972 recently demonstrated robust efficacy in a 12-week, Phase 2 study in T2DM, reducing HbA1c by 1.2% from baseline, with a safety profile more favorable than other GRAs at this stage of development. LGD-6972 is structurally distinct from other small molecule GRAs, containing a sulphonic acid tail (SAT) rather than a carboxylic acid tail (CAT), potentially explaining this finding. We performed computational modeling of LGD-6972 bound to the glucagon receptor (GCGR) and compared it to x-ray crystallography data with receptor bound MK-0893. The SAT of LGD-6972 creates a larger pocket between helix 6 and 7 in the GCGR transmembrane domain, and alters the orientation of helix 8. Activity of LGD-6972, MK-0893 and other SAT and CAT GRAs were examined in cellular models of glucagon-stimulated cAMP accumulation, β-arrestin recruitment, and receptor internalization. All tested GRAs were full (>95%) antagonists of cAMP accumulation. CAT GRAs were full antagonists of β-arrestin (≥96%) and receptor internalization (≥94%). In contrast, SAT GRAs had reduced antagonism of β-arrestin (58-87%) and receptor internalization (6-52%). The relative potency was also altered with greater potency for CAT GRAs on β-arrestin vs. cAMP, whereas the opposite was true for SAT GRAs. Furthermore, a LGD-6972 analog with a CAT and a MK-0893 analog with a SAT displayed CAT- and SAT-like activity, respectively, indicating a clear structure-activity relationship exists, with LGD-6972 displaying receptor-biased signaling. To further investigate the potential differences among compounds, primary human hepatocytes were treated with glucagon or glucagon with MK-0893 or LGD-6972 and gene expression was analyzed by microarray. Broad differences between glucagon and the GRAs were observed and future studies will investigate the effects of SAT and CAT compounds. Disclosure E.G. Vajda: Employee; Self; Ligand Pharmaceuticals, Inc. L. Zhi: Employee; Self; Ligand Pharmaceuticals, Inc. K. Marschke: Employee; Self; Ligand Pharmaceuticals, Inc..

Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW5,19ALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific 2H- and 15N-labeled residues. GW5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as 2H quadrupolar or 15N-1H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW5,19ALP23, the modified GW4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the 2H and 15N NMR signals confirm that the extent of dynamic averaging, particularly rotational “slippage” about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional 2H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment.

Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR.

NMR approaches using nucleotide-specific deuterium labeling schemes have enabled structural studies of biologically relevant RNAs of increasing size and complexity. Although local structure is well-determined using these methods, definition of global structural features, including relative orientations of independent helices, remains a challenge. Residual dipolar couplings, a potential source of orientation information, have not been obtainable for large RNAs due to poor sensitivity resulting from rapid heteronuclear signal decay. Here we report a novel multiple quantum NMR method for RDC determination that employs flip angle variation rather than a coupling evolution period. The accuracy of the method and its utility for establishing interhelical orientations are demonstrated for a 36-nucleotide RNA, for which comparative data could be obtained. Applied to a 78 kDa Rev response element from the HIV-1 virus, which has an effective rotational correlation time of ca. 160 ns, the method yields sensitivity gains of an order of magnitude or greater over existing approaches. Solution-state access to structural organization in RNAs of at least 230 nucleotides is now possible.

Crystallization and structure analysis of the core motif of the Pks13 acyltransferase domain from Mycobacterium tuberculosis.

Type I polyketide synthase 13 (Pks13) is involved in the final step of the biosynthesis of mycolic acid in Mycobacterium tuberculosis. Recent articles have reported that Pks13 is an essential enzyme in the mycolic acid biosynthesis pathway, and it has been deeply studied as a drug target in Tuberculosis. We report a high-resolution structure of the acyltransferase (AT) domain of Pks13 at 2.59 Å resolution. Structural comparison with the full-length AT domain (PDB code, 3TZW, and 3TZZ) reveals a different orientation of the C-terminal helix and rearrangement of some conserved residues.