Predominant Conformation Research Articles

Conformation-Specific Infrared and Ultraviolet Spectroscopy of Cold [YAPAA+H]+ and [YGPAA+H]+ Ions: A Stereochemical "Twist" on the β-Hairpin Turn.

Incorporation of the unnatural d-proline (DP) stereoisomer into a polypeptide sequence is a typical strategy to encourage formation of β-hairpin loops because natural sequences are often unstructured in solution. Using conformation-specific IR and UV spectroscopy of cold (≈10 K) gas-phase ions, we probe the inherent conformational preferences of the DP and LP diastereomers in the protonated peptide [YAPAA+H]+, where only intramolecular interactions are possible. Consistent with the solution-phase studies, one of the conformers of [YADPAA+H]+ is folded into a charge-stabilized β-hairpin turn. However, a second predominant conformer family containing two sequential γ-turns is also identified, with similar energetic stability. A single conformational isomer of the LP diastereomer, [YALPAA+H]+, is found and assigned to a structure that is not the anticipated "mirror image" β-turn. Instead, the LP stereocenter promotes a cis-alanine-proline amide bond. The assigned structures contain clues that the preference of the DP diastereomer to support a trans-amide bond and the proclivity of LP for a cis-amide bond is sterically driven and can be reversed by substituting glycine for alanine in position 2, forming [YGLPAA+H]+. These results provide a basis for understanding the residue-specific and stereospecific alterations in the potential energy surface that underlie these changing preferences, providing insights to the origin of β-hairpin formation.

Facile synthesis, biological evaluation and molecular docking studies of novel substituted azole derivatives

In this study, we synthesized the series of novel azole derivatives and evaluated for enzyme inhibition assays, corresponding kinetic analysis and molecular modeling. Among the investigated bioassays, the oxadiazole derivatives 4a-k were found potent α-glucosidase inhibitors while the Schiff base derivatives 7a-k exhibited considerable potential toward urease inhibition. The inhibition kinetics for the most active compounds were analyzed by the Lineweaver–Burk plots to investigate the possible binding modes of the synthesized compounds toward the tested proteins. Moreover, the detailed docking studies were performed on the synthesized library of 4a-k and 7a-k to study the molecular interaction and binding mode in the active site of the modeled yeast α-glucosidase and Jack Bean Urease, respectively. It could be inferred from docking results that theoretical studies are in close agreement to that of the experimental results. The structure of one of the compound 7k was characterized by the single crystal X-ray diffraction analysis in order to find out the predominant conformation of the molecules.

Accurate Determination of the RNA Three-Way Junctions Via Single-Molecule High-Precision Fret Measurements

In nature RNA helices are often linked via various junctions and bulges that determine overall 3D structure which are used as building blocks and functional components in nanotechnology applications. Hence visualization of RNA conformations is crucial for understanding their biological functions. Förster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling can be used to determine the structure of these junctions. Multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting (eTCSPC) measurements were applied to perform FRET study on systematic series of 6 RNA three- way-junctions (3WJs) derived from the hairpin ribozyme. Bulge and sequence variations were considered as dominant factors influencing junction, two of which have bulge (two and five unpaired nucleotides) in the junction region. We have generated a database of overall 252 different RNA 3WJs labeled with Alexa488 and Cy5 fluorescent dyes. The analysis toolkit included probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation. We detected only one predominant conformer for RNAs 3WJ. Furthermore we report that bulges in the junction determine orientation and rotation of helices, inducing coaxial stacking. We provide a geometrical description of the junction geometry and our results show that small changes in the sequence make dramatic changes in RNA 3WJ tertiary structures which are expected to have significant impact on the functionality. As the accuracy of FRET restrained modeling depends on the correct description of the dye behavior, we characterized the position dependent fluorescent properties of the coupled dyes and statistically analyzed the systematic deviations of the calculated FRET distances from the model generated distances. This allowed us to identify the problematic labeling positions. Thus we could improve the accuracy of the FRET method by careful selection of labeling sites.

Catalytic asymmetric synthesis of 2,3,3,3-tetrafluoro-2-methyl-1-arylpropan-1-amines as useful building blocks for SAR-studies

The first protocol for the asymmetric synthesis of 2,3,3,3-tetrafluoro-2-methyl-1-arylpropan-1-amines which function as fluorinated surrogates for α-isopropylbenzylamines in SAR-studies is presented herein. Crucial for the successful synthesis was the application of a recently developed direct catalytic asymmetric Mannich-type reaction of fluorinated amide for the key bond-forming step. The utility of this versatile protocol was demonstrated by the synthesis of fluorinated analogues of a T-type selective Ca2+ channel blocker and a prolylcarboxypeptidase inhibitor. The predominant conformation of their fluorinated analogues was investigated by X-ray crystallography and NMR spectroscopy which revealed a strong influence of a fluorine mediated gauche effect.

Conformational NMR Study of Bistriazolyl Anion Receptors

Conformational features of pyridine- and pyrimidine-based bistriazolyl anion receptors dissolved in acetonitrile-d3 were assessed by multidimensional, heteronuclear NMR spectroscopy. NOESY correlation signals suggested preorganization of both host molecules in solution in the absence of anions. In addition, only a single set of signals was observed in the 1H NMR spectra, which suggested a symmetrical conformation of anion receptors or their conformational exchange that is fast on the NMR time-scale. Furthermore, the predominant conformations of the pyridine- and pyrimidine-based anion receptors are preserved upon addition of chloride, bromide, and acetate anions. Chemical shift changes observed upon addition of anions showed that the NH (thio)urea and triazole protons are involved in anion-receptor interactions through hydrogen bonding.

Vibrational Sum-Frequency Generation Study of Morpholine at Air–Liquid and Air–Solution Interfaces

The structure and orientation of interfacial morpholine molecules have been investigated, using vibrational sum-frequency generation (VSFG) spectroscopy—a nonlinear surface specific technique. The VSFG spectra with SSP and PPP polarizations have been measured in the CH (2800–3000 cm–1) and the OH (3000–3750 cm–1) stretch regions at the air–morpholine and air–solution interfaces. The vibrational frequencies in the CH stretch region could be observed in VSFG spectra, implying presence of morpholine molecules at the interfaces with net polar orientation. The intensities of the CH stretch bands get enhanced at the air–solution interface of morpholine solution in millimolar concentration, in comparison to that at the air–morpholine interface which is attributed to increase in polar orientation of interfacial morpholine molecules induced by water molecules. In pure morpholine, the most predominant conformation of molecules is equatorial chair, both in the bulk and at the air–morpholine interface. But in aqueous solution of morpholine, the contribution from axial chair conformer is known to increase. This effect, and also a probable change from the chair to the boat/twist boat conformation at the air–solution interface, may contribute to the enhanced intensity of VSFG peaks of CH stretch bands in solution. The VSFG intensities of the OH stretching frequencies of interfacial water molecules are also enhanced in the presence of morpholine, suggesting an increase in net polar orientation of water molecules induced by morpholine molecules. The VSFG spectra were also measured in the presence of 300 mM HCl, which showed indications of protonation of the interfacial morpholine molecules. Addition of HCl to aqueous solution of morpholine alters the orientation of interfacial water molecules significantly, and the enhanced VSFG intensities in the OH region induced by morpholine molecules are almost completely obliterated. The result suggests that orientation of interfacial water molecules in the presence of HCl gets random. However, the effects of HCl in the CH region of the VSFG spectra differ for different stretching bands. In presence of readily ionizable HCl molecules, a large number of ions are generated, which are probably responsible for changing the surface orientation of both water and morpholine molecules.

α-Aminoxy Oligopeptides: Synthesis, Secondary Structure, and Cytotoxicity of a New Class of Anticancer Foldamers.

α-Aminoxy peptides are peptidomimetic foldamers with high proteolytic and conformational stability. To gain an improved synthetic access to α-aminoxy oligopeptides we used a straightforward combination of solution- and solid-phase-supported methods and obtained oligomers that showed a remarkable anticancer activity against a panel of cancer cell lines. We solved the first X-ray crystal structure of an α-aminoxy peptide with multiple turns around the helical axis. The crystal structure revealed a right-handed 28 -helical conformation with precisely two residues per turn and a helical pitch of 5.8 Å. By 2D ROESY experiments, molecular dynamics simulations, and CD spectroscopy we were able to identify the 28 -helix as the predominant conformation in organic solvents. In aqueous solution, the α-aminoxy peptides exist in the 28 -helical conformation at acidic pH, but exhibit remarkable changes in the secondary structure with increasing pH. The most cytotoxic α-aminoxy peptides have an increased propensity to take up a 28 -helical conformation in the presence of a model membrane. This indicates a correlation between the 28 -helical conformation and the membranolytic activity observed in mode of action studies, thereby providing novel insights in the folding properties and the biological activity of α-aminoxy peptides.

Process Development for Separation of Conformers from Derivatives of Resorcin[4]arenes and Pyrogallol[4]arenes.

Macrocyclic compounds, such as resorcin[4]arenes and pyrogallol[4]arenes, have proven to be useful building blocks in the construction of supramolecular organic frameworks (SOFs) because of their unique bowl-like shape and ability to interact through variety of intermolecular interactions. Herein, we report the synthesis and crystal structures of two functionalized resorcin[4]arenes and pyroagllol[4]arenes, 4-hydroxyphenylresorcin[4]arenes, and 4-hydroxyphenylpyrogallol[4]arenes. These phenyl-functionalized macrocycles usually have different conformers, such as cone, boat, chair, saddle, and diamond. The successful separation of predominant conformers from the crude product was carried out with solvent-extraction technique. The shape and molecular arrangement of these conformers in the individual crystal structure was verified with single-crystal X-ray diffraction studies.

Determination of the Absolute Configurations of Chiral Drugs Using Chiroptical Spectroscopy.

Chiroptical spectroscopy has emerged as a promising tool for the determination of absolute configurations and predominant conformations of chiral molecules in academic laboratories. This promise has led to the adaption of chiroptical spectroscopic methods as valuable tools in chiral drug discovery research programs of the pharmaceutical industry. Most major pharmaceutical companies have invested in in-house chiroptical spectroscopy applications and reported successful outcomes. In the context of continuously increasing applications of chiroptical spectroscopy for chiral molecular structure determination, a review of recent developments and applications for chiral drugs is presented in this manuscript.

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1.

The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c in intracellular movements of this critical slit diaphragm protein. Using small angle X-ray scattering studies, models of predominant solution conformation of unliganded full-length Myo1c and Myo1c bound to Neph1 were constructed. The resulting structures show an extended S-shaped Myo1c with Neph1 attached to its C-terminal tail. Importantly, binding of Neph1 did not induce a significant shape change in Myo1c, indicating this as a spontaneous process or event. Analysis of interaction surfaces led to the identification of a critical residue in Neph1 involved in binding to Myo1c. Indeed, a point mutant from this site abolished interaction between Neph1 and Myo1c when tested in the in vitro and in live-cell binding assays. Live-cell imaging, including fluorescence recovery after photobleaching, provided further support for the role of Myo1c in intracellular vesicular movement of Neph1 and its turnover at the membrane.

Eight new crystal structures of 5-(hydroxymethyl)uracil, 5-carboxyuracil and 5-carboxy-2-thiouracil: insights into the hydrogen-bonded networks and the predominant conformations of the C5-bound residues.

In order to examine the preferred hydrogen-bonding pattern of various uracil derivatives, namely 5-(hydroxymethyl)uracil, 5-carboxyuracil and 5-carboxy-2-thiouracil, and for a conformational study, crystallization experiments yielded eight different structures: 5-(hydroxymethyl)uracil, C5H6N2O3, (I), 5-carboxyuracil-N,N-dimethylformamide (1/1), C5H4N2O4·C3H7NO, (II), 5-carboxyuracil-dimethyl sulfoxide (1/1), C5H4N2O4·C2H6OS, (III), 5-carboxyuracil-N,N-dimethylacetamide (1/1), C5H4N2O4·C4H9NO, (IV), 5-carboxy-2-thiouracil-N,N-dimethylformamide (1/1), C5H4N2O3S·C3H7NO, (V), 5-carboxy-2-thiouracil-dimethyl sulfoxide (1/1), C5H4N2O3S·C2H6OS, (VI), 5-carboxy-2-thiouracil-1,4-dioxane (2/3), 2C5H4N2O3S·3C6H12O3, (VII), and 5-carboxy-2-thiouracil, C10H8N4O6S2, (VIII). While the six solvated structures, i.e. (II)-(VII), contain intramolecular S(6) O-H...O hydrogen-bond motifs between the carboxy and carbonyl groups, the usually favoured R2(2)(8) pattern between two carboxy groups is formed in the solvent-free structure, i.e. (VIII). Further R2(2)(8) hydrogen-bond motifs involving either two N-H...O or two N-H...S hydrogen bonds were observed in three crystal structures, namely (I), (IV) and (VIII). In all eight structures, the residue at the ring 5-position shows a coplanar arrangement with respect to the pyrimidine ring which is in agreement with a search of the Cambridge Structural Database for six-membered cyclic compounds containing a carboxy group. The search confirmed that coplanarity between the carboxy group and the cyclic residue is strongly favoured.

Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.

Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies.

Accurate Determination of the RNA Junctions via Single-Molecule High-Precision FRET Measurements

Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing the biomolecular structure. We apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements (eTCSPC) to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) which are derived from the hairpin ribozyme.Overall 283 FRET pairs were measured with single-molecule MFD and analyzed with the analysis toolkit [1] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation.In order to study the influence of the junction on the RNA structure we studied the functional junction part with prolonged helices. Bulge and sequence variations were considered as dominant factors influencing junction conformations for RNA 3WJ. Six different RNA 3WJ with different sequences were studied, two of which have two and five unpaired nucleotides in the junction region.We found three different conformers for RNA 4WJ. However RNAs 3WJ have only one predominant conformer. Furthermore we report that bulges in the junction region determine orientation and rotation of helices, inducing coaxial stacking. Noteworthy the stacked helices are different for the 3WJs with different bulges. Our results show that small changes in the sequence make dramatic changes in RNA 3WJ tertiary structures which are expected to have significant impact on the functionality.[1] Kalinin, S. et al, Nature Methods, 9, 1218-1225 (2012).

A comparative study of the vibrational spectra of the anticancer drug melphalan and its fundamental molecules 3-phenylpropionic acid and l-phenylalanine

The structural stability and the vibrational spectra of the anticancer drug melphalan and its parent compounds 3-phenylpropionic acid and l-phenylalanine were investigated by the DFT B3LYP/6-311G** calculations. Melphalan and its fundamental compounds were predicted to exist predominantly in non-planar structures. The vibrational frequencies of the low energy structures of melphalan, 3-phenylpropionic acid, and phenylalanine were computed at the DFT B3LYP level of theory. Complete vibrational assignments of the normal modes of melphalan, 3-phenylpropionic acid, and phenylalanine were provided by combined theoretical and experimental data of the molecules. The experimental infrared spectra of phenylalanine and melphalan show a significantly different pattern of the CO stretching mode as compared to those of normal carboxylic acids. A comparison of the 3700–2000 cm−1 infrared spectral region of the three molecules suggests the presence of similar intermolecular H-bonding in their condensed phases. The observed infrared and Raman spectra are consistent with the presence of one predominant melphalan conformation at room temperature.

Supersaturation-dependent polymorphic outcome and transformation rate ofl-glutamic acid

A link between the predominant conformer in supersaturatedl-Glu aqueous solution and the polymorphic outcome is built using FTIR.

Synthesis and conformational analysis of 6-[N,N-bis(dihydroxyphosphorylmethyl)amino]methyl-2-hydroxy-2-oxido-1,4,2-oxazaphosphorinane-4-methylphosphonic acid by NMR spectroscopy

The Kabachnik—Fields methylphosphorylation of 1,3-diaminopropan-2-ol affords a mixture of 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetrakismethylphosphonic acid and its intramolecular cyclic ester. Subsequent heating of this mixture led to the thermal dehydration of the acid with the 1,4,2-oxazaphosphorinane ring closure and the formation of 6-[N,N-bis(dihydroxyphosphorylmethyl)amino]methyl-2-hydroxy-2-oxido-1,4,2-oxazaphosphorinane4-methylphosphonic acid. A predominant chair conformation of the formed six-membered heterocycle was inferred from the data of 2D homonuclear (1H, 1H; J-resolved) and heteronuclear (1H, 13C; HSQC, HMBC) NMR correlation spectra.

Cholesterol-Dependent Conformational Exchange of the C-Terminal Domain of the Influenza A M2 Protein.

The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol-dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry-related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol-dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all of our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding.

Conformational diversity of single-stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases.

Repetitive extragenic palindrome (REP)—associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5′-G and 3′-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 585–596, 2015.

Phospholipid-driven differences determine the action of the synthetic antimicrobial peptide OP-145 on Gram-positive bacterial and mammalian membrane model systems

OP-145, a synthetic antimicrobial peptide developed from a screen of the human cathelicidin LL-37, displays strong antibacterial activities and is — at considerably higher concentrations — lytic to human cells. To obtain more insight into its actions, we investigated the interactions between OP-145 and liposomes composed of phosphatidylglycerol (PG) and phosphatidylcholine (PC), resembling bacterial and mammalian membranes, respectively. Circular dichroism analyses of OP-145 demonstrated a predominant α-helical conformation in the presence of both membrane mimics, indicating that the different membrane-perturbation mechanisms are not due to different secondary structures. Membrane thinning and formation of quasi-interdigitated lipid–peptide structures was observed in PG bilayers, while OP-145 led to disintegration of PC liposomes into disk-like micelles and bilayer sheets. Although OP-145 was capable of binding lipoteichoic acid and peptidoglycan, the presence of these bacterial cell wall components did not retain OP-145 and hence did not interfere with the activity of the peptide toward PG membranes. Furthermore, physiological Ca++ concentrations did neither influence the membrane activity of OP-145 in model systems nor the killing of Staphylococcus aureus. However, addition of OP-145 at physiological Ca++-concentrations to PG membranes, but not PC membranes, resulted in the formation of elongated enrolled structures similar to cochleate-like structures. In summary, phospholipid-driven differences in incorporation of OP-145 into the lipid bilayers govern the membrane activity of the peptide on bacterial and mammalian membrane mimics.

Successive Stages of Amyloid-β Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization.

Self-assembly of amyloid-β (Aβ) peptides in human brain tissue leads to neurodegeneration in Alzheimer's disease (AD). Amyloid fibrils, whose structures have been extensively characterized by solid state nuclear magnetic resonance (ssNMR) and other methods, are the thermodynamic end point of Aβ self-assembly. Oligomeric and protofibrillar assemblies, whose structures are less well-understood, are also observed as intermediates in the assembly process in vitro and have been implicated as important neurotoxic species in AD. We report experiments in which the structural evolution of 40-residue Aβ (Aβ40) is monitored by ssNMR measurements on frozen solutions prepared at four successive stages of the self-assembly process. Measurements on transient intermediates are enabled by ssNMR signal enhancements from dynamic nuclear polarization (DNP) at temperatures below 30 K. DNP-enhanced ssNMR data reveal a monotonic increase in conformational order from an initial state comprised primarily of monomers and small oligomers in solution at high pH, to larger oligomers near neutral pH, to metastable protofibrils, and finally to fibrils. Surprisingly, the predominant molecular conformation, indicated by (13)C NMR chemical shifts and by side chain contacts between F19 and L34 residues, is qualitatively similar at all stages. However, the in-register parallel β-sheet supramolecular structure, indicated by intermolecular (13)C spin polarization transfers, does not develop before the fibril stage. This work represents the first application of DNP-enhanced ssNMR to the characterization of peptide or protein self-assembly intermediates.