Comparative Studies of the Electronic Structure of 2-Chloro-2′-Deoxyadenosine Studied by 35CI-NQR Spectroscopy and Quantum Chemical Calculations

Abstract

Chlorine-35 NQR spectroscopy and quantum chemical calculations have been applied to provide detailed information on the structure and conformation of 2-chloro- 2′-deoxyadenosine and 2-chloroadenosine. The Gaussian package was used and the calculations were performed at the B3LYP level of the theory in the 6-31G? basis set.     

In Situ Structural Studies of Anabaena Sensory Rhodopsin in the E.?coli Membrane

Magic-angle spinning nuclear magnetic resonance is well suited for the study of membrane proteins in the nativelike lipid environment. However, the natural cellular membrane is invariably more complex than the proteoliposomes most often used for solid-state NMR (SSNMR) studies, and differences may affect the structure and dynamics of the proteins under examination. In this work we use SSNMR and other biochemical and biophysical methods to probe the structure of a seven-transmembrane helical photoreceptor, Anabaena sensory rhodopsin (ASR), prepared in the Escherichia coli inner membrane, and compare it to that in a bilayer formed by DMPC/DMPA lipids. We find that ASR is organized into trimers in both environments but forms two-dimensional crystal lattices of different symmetries. It favors hexagonal packing in liposomes, but may form a square lattice in the E. coli membrane. To examine possible changes in structure site-specifically, we perform two- and three-dimensional SSNMR experiments and analyze the differences in chemical shifts and peak intensities. Overall, this analysis reveals that the structure of ASR is largely conserved in the inner membrane of E. coli, with many of the important structural features of rhodopsins previously observed in ASR in proteoliposomes being preserved. Small, site-specific perturbations in protein structure that occur as a result of the membrane changes indicate that the protein can subtly adapt to its environment without large structural rearrangement.     

Decay Kinetics of Tyrosine Radical (YZ •) in Chloride Anion-Depleted Photosystem 2 Studied by Time-Resolved EPR

The decay of tyrosine cation radical was found to be biphasic at 253 K. The fast phase corresponds to the Y_Z component while the slow phase corresponds to the tyrosine D radical (Y_D ) component. At 253 K, the t_1/2 value was 28.6 s for the fast phase and 190.7 s for the slow phase. The fast phase is attributed to the recombination of charges between Y_Z and Q_A ^–. The activation energy for the reaction of Y_Z with Q_A ^– between 253 and 293 K was 48 kJ mol^–1 in Cl^–-depleted photosystem 2 (PS2) membranes. Both the decay rate and the amplitude of the PAR -induced signal of Y_Z were affected by addition of chloride anion. Change in the decay rate and the amplitude of the PAR-induced signal of Y_Z was observed when other anions like Br^–, I^–, F, HCO₃ ^–, NO₃ ^–, PO₄ ^3– were substituted in the Cl^–-depleted PS2.     

Interaction of Pyrylium Dye with Self-Complementary DNA Oligomer as Studied by H NMR Spectroscopy‡

Abstract

Interaction of 2-methyl-4,6-bis-(4-N,N-dimethylaminophenyl) pyrylium salt (P2) with [d(CGACGTCG)]₂ was investigated by H NMR spectroscopy. The aromatic signals of P2 and the oligomer were shifted to the upfield by forming the complex, and intermolecular NOEs were also observed between P2 and the terminal CpG base steps but not between P2 and the central CpG. These results indicate that P2 binds to the weakly stacking CpG steps in an intercalation manner.

     

Sedimentation of Bovine Rhodopsin—Digitonin Micelles

RHODOPSIN, the photo-sensitive pigment of vertebrate vision receptors, consists of the lipoprotein opsin bound to the 11-cis isomer of retinal. Light isomerizes the 11-cis configuration to the all-trans, which makes the pigment unstable, leading eventually to the dissociation of the retinal from the lipoprotein. The belief that these dark steps involve conformational changes in the lipoprotein moiety stems from spectroscopic measurements which show the disappearance of lipoprotein-chromophore interactions and from kinetic and thermodynamic considerations^1–5, but more direct evidence has come from the changes in circular dichroism and optical rotatory dispersion which occur with bleaching^6–9. There is also an increase of Stokes radius on bleaching¹¹.     

The Structure of 5-Cyclohexyl-2′-Deoxyuridine as Studied by X-Ray Crystallography and NMR Spectroscopy

Abstract

5-Cyclohexyl-2′-deoxyuridine (I) is an example of a 5-substituted pyrimidine 2′-deoxynucleoside which exhibits no antiviral activity and which is not a substrate for either cellular or viral (herpes) kinases. Despite the fact that a cursory inspection of NMR spectra of the compound, taken in DMSO-d ₆ solution, suggested that the compound had a normal conformation, we here show that in the crystal and in aqueous solution (analysed by 2D NMR techniques), the conformation of this nucleoside has a syn-glycosidic and C4′-exo (₄E) sugar pucker conformation.     

Characteristics of Relationships Between Structure of Gluten Proteins and Dough Rheology – Influence of Dietary Fibres Studied by FT-Raman Spectroscopy

The aim of this research was to study which kind of conformational changes in gluten proteins were induced by addition of four dietary fibre (apple-cranberry, cacao, carob and oat) by using FT-Raman spectroscopy and to find relationships between conformational changes and rheological behaviour of bread dough in mixing and extensional tests. Structural studies showed that all fibres induced formation of β-like structures between two protein molecules (pseudo-β-sheets) with the band at 1616 cm^?1 in the Raman spectrum. According to Principal Component Analysis, the strongest dependence was between changes in gluten structure and two extensographic parameters (resistance to extension and extensibility). Resistance to extension was positively correlated with content of α-helix and pseudo-β-sheets, while a negative correlation was observed between the parameter and content of β-sheets and β-turns. Gauche-gauche-gauche conformation of disulphide bridges and ability of tyrosine residues to hydrogen bonds creation improved mixing properties as stability of dough.     

Micro-Heterogeneity and Micro-Rheological Properties of High-Viscosity Barley β-Glucan Solutions Studied by Diffusing Wave Spectroscopy (DWS)

Soluble fiber β-glucan is one of the key dietary materials in healthy food products known for reducing serum cholesterol levels. The micro-structural heterogeneity and micro-rheology of high-viscosity barley β-glucan solutions were investigated by the diffusing wave spectroscopy (DWS) technology. By comparing the mean-square displacement (MSD) of the microspheres imbedded in eight concentrations of β-glucan solutions, we found that the solutions exhibited nearly homogeneous behavior at ≤0.1 %, but the material showed a clear degree of heterogeneity at ≥0.25 %. Micro-rheology investigation revealed that β-glucan solutions displayed nearly perfect viscous behavior at ≤0.1 %, but the property changed into viscoelastic one at ≥0.25 %. The magnitude of high-frequency viscoelastic moduli for the 0.25 % - 0.75 % β-glucan solutions can be characterized by ?G*? ∝ ω^3/4, which is the semi-flexible polymer behavior. However, the magnitude of high-frequency viscoelastic moduli (?G*?) for the 1.0 % - 1.25 % β-glucan solutions is proportional to ω^1/2, which is the flexible polymer behavior. All micro-structural heterogeneity and micro-rheological property shifts occurred in relatively small concentration ranges.     

Membrane Permeabilization by Oligomeric α-Synuclein: In Search of the Mechanism

Background

The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinson''s disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory.

Methodology/Principal Findings

Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general.

Conclusions/Significance

The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric α-synuclein species.     

Group-Recognition Ability of Derivatized β-Cyclodextrin as Studied by the ESR Spin Probing Technique

The effect of modifying the entrance face of β-cyclodextrin on the inclusion of various functional groups in aminoxyl spin probes was determined by the use of heptakis (2,6-O-dimethyl)-β-cyclodextrin and heptakis (2,3,6-O-triacetyl)-β-cyclodextrin. The methylation of six of the secondary hydroxyl groups on the wider end of β-cyclodextrin has a variable effect on the association constant of inclusion depending on the nature of the included group. The role of the methoxy group on the rim is discussed on the basis of the thermodynamic data derived from association constants. Contrary to the unmodified β-cyclodextrin the pH of the solution does not influence the inclusion behaviour.     

Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy

In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 μm^?2, respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.     

Time-Resolved Surface-Enhanced IR-Absorption Spectroscopy of Direct Electron Transfer to Cytochrome c Oxidase from R.?sphaeroides

The aggregation of α-synuclein is thought to play a role in the death of dopamine neurons in Parkinson’s disease (PD). Alpha-synuclein transitions itself through an aggregation pathway consisting of pathogenic species referred to as protofibrils (or oligomer), which ultimately convert to mature fibrils. The structural heterogeneity and instability of protofibrils has significantly impeded advance related to the understanding of their structural characteristics and the amyloid aggregation mystery. Here, we report, to our knowledge for the first time, on α-synuclein protofibril structural characteristics with cryo-electron microscopy. Statistical analysis of annular protofibrils revealed a constant wall thickness as a common feature. The visualization of the assembly steps enabled us to propose a novel, to our knowledge, mechanisms for α-synuclein aggregation involving ring-opening and protofibril-protofibril interaction events. The ion channel-like protofibrils and their membrane permeability have also been found in other amyloid diseases, suggesting a common molecular mechanism of pathological aggregation. Our direct visualization of the aggregation pathway of α-synuclein opens up fresh opportunities to advance the understanding of protein aggregation mechanisms relevant to many amyloid diseases. In turn, this information would enable the development of additional therapeutic strategies aimed at suppressing toxic protofibrils of amyloid proteins involved in neurological disorders.     

The Z-Conformation of Poly(dA-dT) · Poly(dA-dT) in Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dA-dT) poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni²⁺ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl₂ in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm″^?1coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm^?1 region characterizing local chemical interactions of the Ni²⁺ ions with the adenien N7 position, iii) lines at about 1483-and 1582 cm^?1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680-and 1733 cm^?1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT) poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

Time-Resolved Cell Culture Assay Analyser (TReCCA Analyser) for the Analysis of On-Line Data: Data Integration—Sensor Correction—Time-Resolved IC50 Determination

Time-resolved cell culture assays circumvent the need to set arbitrary end-points and reveal the dynamics of quality controlled experiments. However, they lead to the generation of large data sets, which can represent a complexity barrier to their use. We therefore developed the Time-Resolved Cell Culture Assay (TReCCA) Analyser program to perform standard cell assay analyses efficiently and make sophisticated in-depth analyses easily available. The functions of the program include data normalising and averaging, as well as smoothing and slope calculation, pin-pointing exact change time points. A time-resolved IC₅₀/EC₅₀ calculation provides a better understanding of drug toxicity over time and a more accurate drug to drug comparison. Finally the logarithmic sensor recalibration function, for sensors with an exponential calibration curve, homogenises the sensor output and enables the detection of low-scale changes. To illustrate the capabilities of the TReCCA Analyser, we performed on-line monitoring of dissolved oxygen in the culture media of the breast cancer cell line MCF-7 treated with different concentrations of the anti-cancer drug Cisplatin. The TReCCA Analyser is freely available at www.uni-heidelberg.de/fakultaeten/biowissenschaften/ipmb/biologie/woelfl/Research.html. By introducing the program, we hope to encourage more systematic use of time-resolved assays and lead researchers to fully exploit their data.     

Independent Regulation of Reovirus Membrane Penetration and Apoptosis by the μ1 ? Domain

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein μ1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of μ1 indicates that the μ1 ϕ domain is sufficient to elicit a cell death response. To evaluate the contribution of the μ1 ϕ domain to the induction of apoptosis following reovirus infection, ϕ mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in ϕ diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in ϕ affect the apoptotic potential of reovirus, suggesting that ϕ initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective ϕ mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the ϕ domain of μ1 plays an important regulatory role in reovirus-induced apoptosis and disease.     

Limitations of Time-Resolved Fluorescence Suggested by Molecular Simulations: Assessing the Dynamics of T?cell Receptor Binding Loops

Time-resolved fluorescence anisotropy (TRFA) has a rich history in evaluating protein dynamics. Yet as often employed, TRFA assumes that the motional properties of a covalently tethered fluorescent probe accurately portray the motional properties of the protein backbone at the probe attachment site. In an extensive survey using TRFA to study the dynamics of the binding loops of a αβ T cell receptor, we observed multiple discrepancies between the TRFA data and previously published results that led us to question this assumption. We thus simulated several of the experimentally probed systems using a protocol that permitted accurate determination of probe and protein time correlation functions. We found excellent agreement in the decays of the experimental and simulated correlation functions. However, the motional properties of the probe were poorly correlated with those of the backbone of both the labeled and unlabeled protein. Our results warrant caution in the interpretation of TRFA data and suggest further studies to ascertain the extent to which probe dynamics reflect those of the protein backbone. Meanwhile, the agreement between experiment and computation validates the use of molecular dynamics simulations as an accurate tool for exploring the molecular motion of T cell receptors and their binding loops.     

Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ(1-40), defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ(1-40) is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.