Intramolecular Conformation Research Articles

Conformational Dynamics, Intramolecular Domain Conformation Signaling, and Activation of Apo-FimD Revealed by Single-Molecule Fluorescence Resonance Energy Transfer Studies.

The chaperone-usher secretion pathway is a conserved bacterial protein secretion system dedicated to the biogenesis of adhesive fibers. Usher, a multidomain-containing outer membrane protein, plays a central role in this process by acting as a molecular machine that recruits different chaperone-subunit complexes, catalyzes subunit polymerization, and forms a channel for secretion of the assembled subunits. While recent crystal structural studies have greatly advanced our understanding of the structure and function of ushers, the overall architecture of the full-length apo-usher, the molecular events that dictate conformational changes in usher during pilus biogenesis, and its activation by the specific chaperone-adhesin complex remain largely elusive. Using single-molecule fluorescence resonance energy transfer studies, we found that the substrate-free usher FimD (apo-FimD) adopts a contracted conformation that is distinct from its substrate-bound states; both the N-terminal domain (NTD) and the C-terminal domain (CTD) of apo-FimD are highly dynamic, and FimD coordinates its domain conformational changes via intramolecular domain conformation signaling. By combining these studies with in vitro photo-cross-linking studies, we further show that only the chaperone-bound adhesin (FimC:FimH) can be transferred to the CTD, dislocates the plug domain, and triggers conformational changes in the remaining FimD domains. Taken together, these studies delineate an overall architecture of the full-length apo-FimD, provide detailed mechanic insight into the activation of apo-FimD, and explain why FimD could adjust its conformational states to perform multiple functions in each cycle of pilus subunit addition and ensure that pilus assembly proceeds progressively in a cellular energy-free environment.

The Hcm-Causing Y235S Cmybpc Mutation Accelerates Contractile Function by Altering C1 Domain Structure

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 235 (Y235S) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments in detergent-skinned myocardium and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type or Y235S cMyBPC (KOWT or KOY235S). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in skinned myocardium isolated from KOY235S hearts revealed hypercontractile behavior compared to KOWT hearts, evidenced by accelerated rates of cross-bridge kinetics and increased Ca2+ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggests that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which could alter C1 domain ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior which ultimately may result in development of cardiac hypertrophy and in vivo cardiac dysfunction.

The HCM-causing Y235S cMyBPC mutation accelerates contractile function by altering C1 domain structure

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 237 in humans (residue 235 in mice) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type (WT) or Y235S cMyBPC (KOY235S). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in detergent-skinned myocardium isolated from KOY235S hearts revealed hypercontractile behavior compared to KOWT hearts, evidenced by accelerated cross-bridge kinetics and increased Ca2+ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggest that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which potentially alters its ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior, which ultimately results in the development of cardiac hypertrophy and in vivo cardiac dysfunction.

Detailed mechanism and kinetics of the reaction of Criegee intermediate CH2OO with HCOOH investigated via infrared identification of conformers of hydroperoxymethyl formate and formic acid anhydride.

The reaction of Criegee intermediate CH2OO with HCOOH is important in atmospheric chemistry, but its mechanism and kinetics are little investigated. We recorded time-resolved infrared absorption spectra of transient species produced upon irradiation at 308 nm of a flowing mixture of CH2I2/O2/N2/HCOOH at 298 K. Bands of CH2OO were observed initially upon irradiation; their decrease was accompanied with the appearance of several bands near 887, 925, 1052, 1115, 1170, 1342, 1391, and 1760 cm-1, assigned to the absorption of hydroperoxymethyl formate [HC(O)OCH2OOH, HPMF], that decreased in intensity at a later period with the appearance of absorption bands of the anti-conformer of formic acid anhydride [anti-(HCO)2O, FAN] near 998, 1101, 1767, and 1821 cm-1. The main contributions of the infrared absorption of HPMF are from an open-form conformer, but small contributions from the intramolecular hydrogen-bonded conformer that absorbs near 1070, 1170, and 1732 cm-1 were identified. Observed infrared spectra of both conformers of HPMF and anti-FAN agree satisfactorily with the anharmonic vibrational wavenumbers and IR intensities predicted with the B3LYP/aug-cc-pVTZ method. We derived a rate coefficient for CH2OO + HCOOH to be k = (1.4 ± 0.3) × 10-10 cm3 molecule-1 s-1 from formation of HPMF. We found also that anti-FAN was produced mainly from the open-form conformer with rate coefficient k = (1460 ± 30) s-1; the intramolecular hydrogen-bonded conformer of HPMF is stable.

X-ray Emission Spectrum of Liquid Ethanol: Origin of Split Peaks.

The X-ray emission spectrum of liquid ethanol was calculated using density functional theory and a semiclassical approximation to the Kramers-Heisenberg formula including core-hole-induced dynamics. Our spectrum agrees well with the experimental spectrum. We found that the intensity ratio between the two peaks at 526 and 527 eV assigned as 10a' and 3a″ depends not only on the hydrogen bonding network around the target molecule but also on the intramolecular conformation. This effect is absent in liquid methanol and demonstrates the high sensitivity of X-ray emission to molecular structure. The dependence of spectral features on hydrogen-bonding as well as on dynamical effects following core excitation are also discussed.

Molecular structure in the solid state by X-ray crystallography and SSNMR and in solution by NMR of two 1,4-diazepines

The crystals of two 1,4-diazepines prepared from curcuminoid β-diketones and ethylenediamine were studied by X-ray crystallography and NMR. Their tautomerism, intramolecular hydrogen bonds and conformation were determined.

Interplay of Intermolecular and Intramolecular Hydrogen Bonds on Complex Formation: The 3-Aminopropanol-Water van der Waals Complex.

This combined experimental and theoretical study answers the question whether the intramolecular hydrogen-bond strength in amino alcohols is dependent on the ring size. For this purpose, the rotational spectrum of the 3-aminopropanol-H2O van der Waals complex was recorded using Fourier-transform microwave spectroscopy and fit to the rotational, quadrupole coupling, and centrifugal distortion constants of the Watson A-reduction Hamiltonian. The experimental results are consistent with an ab initio conformation calculated at the MP2/6-311++G(d,p) level that involves the lowest energy 3-aminopropanol monomer and consists of a hydrogen bonding network. The calculated global minimum ab initio complex however comprises a higher energy monomer conformation of 3-aminopropanol. Upon complex formation with water, the O-H····N intramolecular hydrogen bond and OCCN backbone conformation of the lower energy monomer remain unchanged, in contrast to 2-aminoethanol. This behavior is consistent with the increasing strength of the intramolecular hydrogen bond of linear amino alcohols as a function of increasing chain length.

Intramolecular versus intermolecular hydrogen bonds in a novel conjugated dimethylamino-benzylidene-amino-2-naphthoic acid Schiff base.

A new compound based on the D-π-A concept, where D=dimethylamino-phenyl and A=naphthoic acid, separated by an imine motif, was designed, synthesized and characterized. The spectral, energetics, and structural characteristics of the compound were studied thoroughly theoretically by density functional theory (DFT) in the gas and aqueous phases and experimentally (steady-state absorption) in aqueous media with various degrees of polarity and hydrogen bonding ability. This compound shows high sensitivity to the polarity, basicity and proton affinity of the environment. Based on DFT, TD-DFT and NBO analysis, the compound exists in the ground-state with both intermolecular and intramolecular hydrogen bond conformations in association with the -COOH, with latter isomer calculated to be more stable. Furthermore, structural changes via intermolecular solute-solvent interactions, dictate electronic modifications and spectral changes. Graphical abstract Acidic and basic sites in DMAMN involved in protonation/deprotonation.

Conformational Effects through Hydrogen Bonding in a Constrained γ-Peptide Template: From Intraresidue Seven-Membered Rings to a Gel-Forming Sheet Structure.

A series of three short oligomers (di-, tri-, and tetramers) of cis-2-(aminomethyl)cyclobutane carboxylic acid, a γ-amino acid featuring a cyclobutane ring constraint, were prepared, and their conformational behavior was examined spectroscopically and by molecular modeling. In dilute solutions, these peptides showed a number of low-energy conformers, including ribbonlike structures pleated around a rarely observed series of intramolecular seven-membered hydrogen bonds. In more concentrated solutions, these interactions defer to an organized supramolecular assembly, leading to thermoreversible organogel formation notably for the tripeptide, which produced fibrillar xerogels. In the solid state, the dipeptide adopted a fully extended conformation featuring a one-dimensional network of intermolecularly H-bonded molecules stacked in an antiparallel sheet alignment. This work provides unique insight into the interplay between inter- and intramolecular H-bonded conformer topologies for the same peptide template.

Reverse Intramolecular Stacking in o‐Xylene Bridge Symmetrical Dimers of 2‐Thiopyridine Derivative: Assessment of the Conformational Stability

AbstractHerein, we report o‐xylene based symmetrical dimers of 2‐thiopyridine derivative for studies of intramolecular stacking and conformational analysis 1‐3. The conformational analyses of all compounds have been carried out in solid, solution and gaseous states. Interestingly, all compounds showed reverse intramolecular stacked conformation between contiguous rings. Among all, compound 3 has shown polymorphism properties that also shown reverse intramolecular stacked conformation. The polymorphic studies have been achieved through single crystal X‐ray diffraction, intermolecular interactions in their crystal packing, overlay diagram, DSC analysis and Hirshfeld surface analysis.

Conformational Ensembles of the Wild-Type and S8C Aβ1-42 Dimers.

We characterized the dimer of the amyloid-β wild-type (WT) peptide, Aβ, of 42 residues and its disulfide-bond-locked double mutant (S8C) by replica exchange molecular dynamics simulations. Aβ dimers are known to be the smallest toxic species in Alzheimer's disease, and the S8C mutant has been shown experimentally to form an exclusive homogeneous and neurotoxic dimer. Our 50 μs all-atom simulations reveal similar secondary structures and collision cross-sections but very different intramolecular and intermolecular conformations upon double S8C mutation. Both dimers are very dynamic with hundreds of free-energy minima that differ from the U-shape and S-shape conformations of the peptides in the fibrils. The only common structural feature, shared by both species with a probability of 4% in WT and 12% in S8C-S8C, is a three-stranded β-sheet spanning the 17-23, 29-36, and 39-41 residues, which does not exist in the Aβ40 WT dimers.

Pressure Tuning Dual Fluorescence of 4-(N,N-Dimethylamino)benzonitrile

The intramolecular charge-transfer (ICT) emission band in the dual fluorescence of the 4-(N,N-dimethylamino)benzonitrile (DMABN) molecular crystal exhibits increase in response to compression up to 10 GPa. On the basis of Raman and angle-dispersive X-ray diffraction (ADXRD) experiments combining with computational studies, the mechanism of this phenomenon could be assigned to the change of the intramolecular geometrical conformation, especially for the decrease of the dihedral angle between the dimethylamino (NMe2) and phenyl moieties. Meanwhile the reduction of excited-state energies and the HOMO–LUMO band gap leads to the redshifts of photoluminescence (PL) spectra and the absorption edge, respectively. Competing with the aggregation caused quenching (ACQ) effect, the planarity of molecular conformation and the slight rotation of the NMe2 group under high pressure both could enhance the ICT process, which will contribute to the revelation of the ICT mechanism and designs of new piezochromic luminescent materials.

Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy

The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson’s disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fibrils varies as a function of ionic strength: fibrils aggregated in low ionic-strength buffers ([NaCl] ≤ 25 mM) have a significantly different structure than fibrils grown in higher ionic-strength buffers. The observations for fibrils aggregated in low-salt buffers are consistent with an extended conformation of αS molecules, forming hydrogen-bonded intermolecular β-sheets that are loosely packed in a parallel fashion. For fibrils aggregated in high-salt buffers (including those prepared in buffers with a physiological salt concentration) the measurements are consistent with αS molecules in a more tightly-packed, antiparallel intramolecular conformation, and suggest a structure characterized by two twisting stacks of approximately five hydrogen-bonded intermolecular β-sheets each. We find evidence that the high-frequency peak in the amide-I spectrum of αS fibrils involves a normal mode that differs fundamentally from the canonical high-frequency antiparallel β-sheet mode. The high sensitivity of the fibril structure to the ionic strength might form the basis of differences in αS-related pathologies.

"Inverted" porphyrins: a distorted adsorption geometry of free-base porphyrins on Cu(111).

Based on density functional theory calculations combined with experimental results, we report and discuss an extremely distorted, "inverted" adsorption geometry of free-base tetraphenylporphyrin on Cu(111). The current findings yield new insights into a well-studied system, shedding light on the peculiar molecule-substrate interaction and the resulting intramolecular conformation.

Regulation of intra- and intermolecular Pt–Pt and π–π interactions of a U-shaped diplatinum complex to achieve pseudo-polymorphic emissions in solution and crystalline states

Pseudo-polymorphic emissions of a U-shaped di-[Pt(NCN)] complex were achieved in crystalline and solution states by regulating the intramolecular conformation (open or closed) and intermolecular stacking mode (with or without Pt–Pt interaction).

Controlling Intramolecular Conformation through Nonbonding Interaction for Soft-Conjugated Materials: Molecular Design and Optoelectronic Properties.

To address the intrinsic contradiction between high optoelectronic properties and good processability in organic π-conjugated molecules, we propose that soft-conjugated molecules (SCMs), conformationally locked by intramolecular nonbonding interactions, can benefit from both nonplanar molecular structures in solution for processing and rigid coplanar structures in the solid state for enhanced optoelectronic properties. Computational results reveal that nonbonding pairs of S···N, N···H, and F···S are strong enough to prevail over thermal fluctuations, steric effects, and other repulsive interactions to force the adjacent aromatic rings to be planar; thus, constructed SCMs display delocalized frontier molecular orbitals with frontier orbital energy levels, band gaps, reorganization energies, and photophyscial properties comparable to those of rigid-conjugated molecules because of their stable planar soft-conjugation at both ground and excited states. The understanding gained from the theoretical investigations of SCMs provides keen insights into construction and modification of soft-conjugations to harmonize the optoelectronic property and processability in conjugated molecules for advanced optoelectronic applications.

Impact of the A2V Mutation on the Heterozygous and Homozygous Aβ1-40 Dimer Structures from Atomistic Simulations.

The A2V mutation was reported to protect from Alzheimer's disease in its heterozygous form and cause an early Alzheimer's disease type dementia in its homozygous form. Experiments showed that the aggregation rate follows the order A2V > WT (wild-type) > A2V-WT. To understand the impact of this mutation, we carried out replica exchange molecular dynamics simulations of Aβ1-40 WT-A2V and A2V-A2V dimers and compared to the WT dimer. Our atomistic simulations reveal that the mean secondary structure remains constant, but there are substantial differences in the intramolecular and intermolecular conformations upon single and double A2V mutation. Upon single mutation, the intrinsic disorder is reduced, the intermolecular potential energies are reduced, the population of intramolecular three-stranded β-sheets is increased, and the number of all α dimer topologies is decreased. Taken together, these results offer an explanation for the reduced aggregation rate of the Aβ1-40 A2V-WT peptides and the protective effect of A2V in heterozygotes.

2H-Tetrakis(3,5-di-tert-butyl)phenylporphyrin on a Cu(110) Surface: Room-Temperature Self-Metalation and Surface-Reconstruction-Facilitated Self-Assembly.

The adsorption behavior of 2H-tetrakis(3,5-di-tert-butyl)phenylporphyrin (2HTTBPP) on Cu(110) and Cu(110)-(2×1)O surfaces have been investigated by using variable-temperature scanning tunneling microscopy (STM) under ultrahigh vacuum conditions. On the bare Cu(110) surface, individual 2HTTBPP molecules are observed. These molecules are immobilized on the surface with a particular orientation with respect to the crystallographic directions of the Cu(110) surface and do not form supramolecular aggregates up to full monolayer coverage. In contrast, a chiral supramolecular structure is formed on the Cu(110)-(2×1)O surface, which is stabilized by van der Waals interactions between the tert-butyl groups of neighboring molecules. These findings are explained by weakened molecule-substrate interactions on the Cu(110)-(2×1)O surface relative to the bare Cu(110) surface. By comparison with the corresponding results of Cu-tetrakis(3,5-di-tert-butyl)phenylporphyrin (CuTTBPP) on Cu(110) and Cu(110)-(2×1)O surfaces, we find that the 2HTTBPP molecules can self-metalate on both surfaces with copper atoms from the substrate at room temperature (RT). The possible origins of the self-metalation reaction at RT are discussed. Finally, peculiar irreversible temperature-dependent switching of the intramolecular conformations of the investigated molecules on the Cu(110) surface was observed and interpreted.

Global and Local Conformational Heterogeniety Governs the Pre-Nucleation Phase in Amyloidogenic Self-Assembly

The formation of amyloid fibrils has been found to be a nucleation-dependent reaction for which the emergence of oligomeric entities is an inevitable and thus presumably, a critical intermediate step in the filamentous growth process. So far it has been not straight-forward to translate the extensive structural knowledge gained for the stable, fibrillar and crystalline states to the soluble oligomers prevalent during the pre-nucleation phase of the multi-step aggregation pathways. In this contribution we investigate the dynamical and structural properties of pre-brillar oligomeric states at atomistic resolution for several amyloidogenic peptide and protein fragments using unbiased explicit solvent molecular dynamics (MD) simulations. We have examined in particular the following questions: Which common secondary and teriary structural characteristics can be found in low molecular weight amyloid oligomers? What role do self-complentary side chain interfaces of intermolecular beta-sheets play in the oligomerization process? We analyzed simulations using multiple force fields in a consensus approach, in addition the results are compared in the light of recent experimental measurements done on model systems for peptide oligomerization. An extensive collision cross section analysis revealed heterogeneous oligomeric aggregates that lack a single, defined tertiary structure during the pre-nucleation phase. To gain insight into the structural organization of the early aggregates spontaneously formed on the microsecond time scale, we probed the substructure of the oligomers and found an increased ordering of intra-molecular conformations and inter-molecular packing motifs with growing aggregate size. We furthermore found that the ensemble of ordered higher-order oligomers sampled a multitude of common beta-structure motifs and was composed of emerging beta-sheet subunit layers. Our results suggest that distinct fibril-like conformations emerge in the oligomeric state and underscore the notion that certain pre-nucleated oligomers serve as a critical intermediate step on-pathway to fibrils.

Spectroscopic and Theoretical Study of the Intramolecular π-Type Hydrogen Bonding and Conformations of 2-Cyclohexen-1-ol.

The infrared and Raman spectra of 2-cyclohexen-1-ol have been recorded and analyzed. The experimental work has been complemented by ab initio and density functional theory computations. The calculations show that in the vapor phase the conformations with the π-type hydrogen bonding are the lowest in energy, and these findings are supported by the experimental spectra, which agree well with the theoretical predictions. The six conformers predicted result from differences between the direction on the ring-twisting angle and the -OH internal rotation angle. The lowest-energy conformer has the hydrogen of the OH group pointing to the middle of the C═C double bond. The other conformers are calculated to be 72 cm(-1) (0.21 kcal/mol) to 401 cm(-1) (1.15 kcal/mol) higher in energy. In the liquid phase, only two conformers can be identified in the spectra, and these correspond to different directions of the ring-twisting.