Backbone Sites Research Articles

15N CSA tensors and 15N–1H dipolar couplings of protein hydrophobic core residues investigated by static solid-state NMR

In this work, we assess the usefulness of static 15N NMR techniques for the determination of the 15N chemical shift anisotropy (CSA) tensor parameters and 15N–1H dipolar splittings in powder protein samples. By using five single labeled samples of the villin headpiece subdomain protein in a hydrated lyophilized powder state, we determine the backbone 15N CSA tensors at two temperatures, 22 and −35°C, in order to get a snapshot of the variability across the residues and as a function of temperature. All sites probed belonged to the hydrophobic core and most of them were part of α-helical regions. The values of the anisotropy (which include the effect of the dynamics) varied between 130 and 156ppm at 22°C, while the values of the asymmetry were in the 0.32–0.082 range. The Leu-75 and Leu-61 backbone sites exhibited high mobility based on the values of their temperature-dependent anisotropy parameters. Under the assumption that most differences stem from dynamics, we obtained the values of the motional order parameters for the 15N backbone sites. While a simple one-dimensional line shape experiment was used for the determination of the 15N CSA parameters, a more advanced approach based on the “magic sandwich” SAMMY pulse sequence (Nevzorov and Opella, 2003) was employed for the determination of the 15N–1H dipolar patterns, which yielded estimates of the dipolar couplings. Accordingly, the motional order parameters for the dipolar interaction were obtained. It was found that the order parameters from the CSA and dipolar measurements are highly correlated, validating that the variability between the residues is governed by the differences in dynamics. The values of the parameters obtained in this work can serve as reference values for developing more advanced magic-angle spinning recoupling techniques for multiple labeled samples.

Enhancement of the thermoelectric figure of merit in DNA-like systems induced by Fano and Dicke effects.

We report a theoretical study highlighting the thermoelectric properties of biological and synthetic DNA molecules. Based on an effective tight-binding model of duplex DNA and by using the nonequilibrium Green's function technique, the thermal conductance, electrical conductance, Seebeck coefficient and thermoelectric figure of merit in the system are numerically calculated by varying the asymmetries of energies and electronic hoppings in the backbone sites to simulate the environmental complications and fluctuations. We find that due to the multiple transport paths in the DNA molecule, the Fano antiresonance occurs, and enhances the Seebeck coefficient and the figure of merit. When the energy difference is produced in every opposite backbone site, the Dicke effect appears. This effect gives rise to a semiconducting-metallic transition, and enhances the thermoelectric efficiency of the DNA molecule remarkably. Moreover, as the Fano antiresonance point is close to the Dicke resonance one, a giant enhancement in the thermoelectric figure of merit in the DNA molecule has been found. These results provide a scenario to obtain effective routes to enhance the thermoelectric efficiency in the DNA molecules, and suggest perspectives for future experiments intending to control the thermoelectric transport in DNA-like nanodevices.

Cross-linked poly(arylene ether ketone) proton exchange membranes sulfonated on polymer backbone, pendant, and cross-linked sites for enhanced proton conductivity

The sulfonated poly(arylene ether ketone) (SPAEK) precursor containing pendant carboxylic groups was synthesized, and used for the production of a series of cross-linked sulfonated poly(arylene ether ketone) membranes with different cross-linking density, using the carboxylic acid reactive groups. In order to obtain high proton conductivity, the sulfonated groups were attached not only on the polymer backbone, but also on the pendant and cross-linked sites. The chemical synthesis of SPAEK was confirmed using 1H-NMR and FT-IR spectroscopy. The proton conductivity, water uptake, mechanical properties, and morphology of the cross-linked membranes (CSPAEK) prepared were investigated, and correlated with the ionic cluster dimension. The use of sulfonated cross-linker had an excellent effect on the overall membrane properties. It enhanced the mechanical properties but noticeably lowered the water uptake, while still maintaining proton conductivity at a high level. Even the CSPAEK membrane with 15% cross-linking degree exhibited higher proton conductivity than Nafion® 117, while demonstrating acceptable water uptake and tensile strength. The ion cluster dimension of the CSPAEK membranes was analyzed using SAXS. The resulting membrane properties were well explained by the SAXS patterns.

Electric field induced localization phenomena in a ladder network with superlattice configuration: Effect of backbone environment

Electric field induced localization properties of a tight-binding ladder network in presence of backbone sites are investigated. Based on Green's function formalism we numerically calculate two-terminal transport together with density of states for different arrangements of atomic sites in the ladder and its backbone. Our results lead to a possibility of getting multiple mobility edges which essentially plays a switching action between a completely opaque to fully or partly conducting region upon the variation of system Fermi energy, and thus, support in fabricating mesoscopic or DNA-based switching devices.

Pore network design: DPD-Monte Carlo study of solvent diffusion dependence on side chain location

Phase separation within water containing polymer membranes is studied by dissipative particle dynamics (DPD). The polymers are composed of hydrophobic backbone A beads to which ([A-C] or [A-A-C-C]) side chains are attached with a hydrophilic (C) end moiety. Water diffusion through the water containing pores is modeled by Monte Carlo (MC) tracer diffusion. Several polymeric architectures are considered which differ in the way side chains are attached along the polymer backbones. In total 120 pore morphologies are stored on file and probed by MC tracer diffusion calculations. For membranes of the same ion exchange capacity diffusion is highest for architectures for which two side chains are branching off from the backbone sites as compared to architectures for which only one side chain is branching off. This conclusion is also obtained from diffusion constants derived from mean squared displacements of water during the DPD simulations. The intrinsic high polymer mobility in conventional DPD results in water diffusivities that are much higher than those obtained from the MC calculations. Assigning higher masses to the polymer beads results in significant decrease in water bead motion.

NMR structures of membrane proteins in phospholipid bilayers.

Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins, especially those accomplished with the proteins in phospholipid bilayer environments where they function.

Dipolar Assisted Assignment Protocol (DAAP) for MAS solid-state NMR of rotationally aligned membrane proteins in phospholipid bilayers

A method for making resonance assignments in magic angle spinning solid-state NMR spectra of membrane proteins that utilizes the range of heteronuclear dipolar coupling frequencies in combination with conventional chemical shift based assignment methods is demonstrated. The Dipolar Assisted Assignment Protocol (DAAP) takes advantage of the rotational alignment of the membrane proteins in liquid crystalline phospholipid bilayers. Improved resolution is obtained by combining the magnetically inequivalent heteronuclear dipolar frequencies with isotropic chemical shift frequencies. Spectra with both dipolar and chemical shift frequency axes assist with resonance assignments. DAAP can be readily extended to three- and four-dimensional experiments and to include both backbone and side chain sites in proteins.

A Combined NMR and Molecular Dynamics Investigation of Sequence Context Effects on Backbone Dynamics of DNA

The mechanisms by which proteins recognize and bind to specific DNA sequences underlie the regulation of basic cell cycle events. Protein recognition sites are frequently regarded in terms of direct readout: as short, rigid sequences that a protein recognizes via base edge contacts. Protein-DNA recognition often operates by indirect readout, in which protein-DNA complexes involve contacts to the phosphate backbone. Dynamic variation of these backbone sites likely plays a large role in binding specificity; one form of dynamic backbone variation is the conformational equilibrium between the BI and BII substates of the canonical B-form of DNA.In this work we perform MD simulations of six DNA sequences, previously characterized using 31P NMR, that contain biologically relevant protein recognition sites with differing flanking sequences. The synergistic use of NMR experiments and MD simulations allows for the rigorous assessment of BI/BII profiles in order to study sequence context effects on the conformational ensemble and dynamics of the DNA backbone. Taken together, our NMR and MD data indicate that sequence context plays a significant role in backbone dynamics; most prominently, A- and T-heavy flanking sequences appear to largely quench backbone dynamics. These data also demonstrate that recent MD force field enhancements are able to successfully reproduce patterns of backbone structure determined by NMR. Moreover, the MD simulations enable investigation of sequence steps that remain unresolved in crowded NMR regions, while the NMR data suggest avenues for future force field improvements.

Colocalization of Fast and Slow Timescale Dynamics in the Allosteric Signaling Protein CheY

It is now widely recognized that dynamics are important to consider for understanding allosteric protein function. However, dynamics occur over a wide range of timescales, and how these different motions relate to one another is not well understood. Here, we report an NMR relaxation study of dynamics over multiple timescales at both backbone and side-chain sites upon an allosteric response to phosphorylation. The response regulator, Escherichia coli CheY, allosterically responds to phosphorylation with a change in dynamics on both the microsecond-to-millisecond (μs-ms) timescale and the picosecond-to-nanosecond (ps-ns) timescale. We observe an apparent decrease and redistribution of μs-ms dynamics upon phosphorylation (and accompanying Mg2+ saturation) of CheY. Additionally, methyl groups with the largest changes in ps-ns dynamics localize to the regions of conformational change measured by μs-ms dynamics. The limited spread of changes in ps-ns dynamics suggests a distinct relationship between motions on the μs-ms and ps-ns timescales in CheY. The allosteric mechanism utilized by CheY highlights the diversity of roles dynamics play in protein function.

Isomer-Selected Photoelectron Spectroscopy of Isolated DNA Oligonucleotides: Phosphate and Nucleobase Deprotonation at High Negative Charge States

Fractionation according to ion mobility and mass-to-charge ratio has been used to select individual isomers of deprotonated DNA oligonucleotide multianions for subsequent isomer-resolved photoelectron spectroscopy (PES) in the gas phase. Isomer-resolved PE spectra have been recorded for tetranucleotides, pentanucleotides, and hexanucleotides. These were studied primarily in their highest accessible negative charge states (3-, 4-, and 5-, respectively), as provided by electrospraying from room temperature solutions. In particular, the PE spectra obtained for pentanucleotide tetraanions show evidence for two coexisting classes of gas-phase isomeric structures. We suggest that these two classes comprise: (i) species with excess electrons localized exclusively at deprotonated phosphate backbone sites and (ii) species with at least one deprotonated base (in addition to several deprotonated phosphates). By permuting the sequence of bases in various [A(5-x)T(x)](4-) and [GT(4)](4-) pentanucleotides, we have established that the second type of isomer is most likely to occur if the deprotonated base is located at the first or last position in the sequence. We have used a combination of molecular mechanics and semiempirical calculations together with a simple electrostatic model to explore the photodetachment mechanism underlying our photoelectron spectra. Comparison of predicted to measured photoelectron spectra suggests that a significant fraction of the detected electrons originates from the DNA bases (both deprotonated and neutral).

Site Specific Interaction of the Polyphenol EGCG with the SEVI Amyloid Precursor Peptide PAP(248–286)

Recently, a 39 amino acid peptide fragment from prostatic acid phosphatase has been isolated from seminal fluid that can enhance infectivity of the HIV virus by up to 4-5 orders of magnitude. PAP(248-286) is effective in enhancing HIV infectivity only when it is aggregated into amyloid fibers termed SEVI. The polyphenol EGCG (epigallocatechin-3-gallate) has been shown to disrupt both SEVI formation and HIV promotion by SEVI, but the mechanism by which it accomplishes this task is unknown. Here, we show that EGCG interacts specifically with the side chains of monomeric PAP(248-286) in two regions (K251-R257 and N269-I277) of primarily charged residues, particularly lysine. The specificity of interaction to these two sites is contrary to previous studies on the interaction of EGCG with other amyloidogenic proteins, which showed the nonspecific interaction of EGCG with exposed backbone sites of unfolded amyloidogenic proteins. This interaction is specific to EGCG as the related gallocatechin (GC) molecule, which shows greatly decreased antiamyloid activity, exhibits minimal interaction with monomeric PAP(248-286). The EGCG binding was shown to occur in two steps, with the initial formation of a weakly bound complex followed by a pH dependent formation of a tightly bound complex. Experiments in which the lysine residues of PAP(248-286) have been chemically modified suggest the tightly bound complex is created by Schiff-base formation with lysine residues. The results of this study could aid in the development of small molecule inhibitors of SEVI and other amyloid proteins.

Quantification of hydroxyl radical-derived oxidation products in peptides containing glycine, alanine, valine, and proline

Proteins are a major target for oxidation due to their abundance and high reactivity. Despite extensive investigation over many years, only limited quantitative data exist on the contributions of different pathways to the oxidation of peptides and proteins. This study was designed to obtain quantitative data on the nature and yields of oxidation products (alcohols, carbonyls, hydroperoxides, fragment species) formed by a prototypic oxidant system (HO •/O 2) on small peptides of limited, but known, amino acid composition. Peptides composed of Gly, Ala, Val, and Pro were examined with particular emphasis on the peptide Val-Gly-Val-Ala-Pro-Gly, a repeat motif in elastin with chemotactic activity and metalloproteinase regulation properties. The data obtained indicate that hydroperoxide formation occurs nonrandomly (Pro > Val > Ala > Gly) with this inversely related to carbonyl yields (both peptide-bound and released). Multiple alcohols are generated at both side-chain and backbone sites. Backbone fragmentation has been characterized at multiple positions, with sites adjacent to Pro residues being of major importance. Summation of the product concentrations provides clear evidence for the occurrence of chain reactions in peptides exposed to HO •/O 2, with the overall product yields exceeding that of the initial HO • generated.

N-Protonated Isomers as Gateways to Peptide Ion Fragmentation

According to the popular "mobile proton model" for peptide ion fragmentation in tandem mass spectrometry, peptide bond cleavage is typically preceded by intramolecular proton transfer from basic sites to an amide nitrogen in the backbone. If the intrinsic barrier to dissociation is the same for all backbone sites, the fragmentation propensity at each amide bond should reflect the stability of the corresponding N-protonated isomer. This hypothesis was tested by using ab initio and force-field computations on several polyalanines and Leu-enkephalin. The results agree acceptably with experimental reports, supporting the hypothesis. It was found that backbone N-protonation is most favorable near the C-terminus. The preference for C-terminal N-protonation, which is stronger for longer polyalanines, may be understood in terms of the well known "helix macrodipole" in the corresponding helical conformations. The opposite stability trend is found for peptides constrained to be linear, which is initially surprising but turns out to be consistent with the reversed direction of the macrodipole in the linear conformation.

Local and Global Dynamics of the G Protein-Coupled Receptor CXCR1

The local and global dynamics of the chemokine receptor CXCR1 are characterized using a combination of solution NMR and solid-state NMR experiments. In isotropic bicelles (q = 0.1), only 13% of the expected number of backbone amide resonances is observed in (1)H/(15)N HSQC solution NMR spectra of uniformly (15)N-labeled samples; extensive deuteration and the use of TROSY made little difference in the 800 MHz spectra. The limited number of observed amide signals is ascribed to mobile backbone sites and assigned to specific residues in the protein; 19 of the signals are from residues at the N-terminus and 25 from residues at the C-terminus. The solution NMR spectra display no evidence of local backbone motions from residues in the transmembrane helices or interhelical loops of CXCR1. This finding is reinforced by comparisons of solid-state NMR spectra of both magnetically aligned and unoriented bilayers containing either full-length or doubly N- and C-terminal truncated CXCR1 constructs. CXCR1 undergoes rapid rotational diffusion about the normal of liquid crystalline phospholipid bilayers; reductions in the frequency span and a change to axial symmetry are observed for both carbonyl carbon and amide nitrogen chemical shift powder patterns of unoriented samples containing (13)C- and (15)N-labeled CXCR1. In contrast, when the phospholipids are in the gel phase, CXCR1 does not undergo rapid global reorientation on the 10(4) Hz time scale defined by the carbonyl carbon and amide nitrogen chemical shift powder patterns.

Influence of backbone on the charge transport properties of G4-DNA molecules: amodel-based calculation

We put forward a model Hamiltonian to describe the influence of backbone energetics oncharge transport through guanine-quadruplex DNA (G4-DNA) molecules. Our analyticalresults show that an energy gap can be produced in the energy spectrum of G4-DNA byhybridization effects between the backbone and the base and by on-site energy difference ofthe backbone from the base. The environmental effects are investigated by introducingdifferent types of disorder into the backbone sites. Our numerical results suggest that thelocalization length of G4-DNA can be significantly enhanced by increasing the backbonedisorder degree when the environment-induced disorder is sufficiently large. There exists abackbone disorder-induced semiconducting–metallic transition in short G4-DNA molecules,where G4-DNA behaves as a semiconductor if the backbone disorder is weak andbehaves as a conductor if the backbone disorder degree surpasses a critical value.

Influence of solitons on the conductance properties of double-stranded deoxyribonucleic acid

A numerical study is presented to investigate the role of solitons in the electronic states of double-stranded DNA (dsDNA) molecule in the metal/DNA/metal system. Based on tight-binding Hamiltonian model and within the framework of a generalized Green’s function technique, we consider a ladder model for poly(dG)-poly(dC) DNA molecule containing M cells with four sites (two base pair sites and two backbone sites) in each cell. In the presence of a sublattice of solitons, our results show that the homogeneous soliton distributions induce the electronic states in the band gap of DNA molecule. In addition, the room temperature current-voltage characteristic of the system shows a linear and ohmic-like behaviour.

Transport properties of poly(GACT)-poly(CTGA) deoxyribonucleic acid: A ladder model approach

In this paper, based on the tight-binding Hamiltonian model and within the framework of a generalized Green’s function technique, the electronic conduction through the poly(GACT)-poly(CTGA) DNA molecule in SWNT/DNA/SWNT structure has been numerically investigated. In a ladder model, we consider DNA as a planar molecule containing M cells and four further sites (two base pair sites and two backbone sites) in each cell, sandwiched between two semi-infinite single-walled carbon nanotubes (SWNT) as the electrodes. Having relied on Landauer formalism, we focussed on studying the current-voltage characteristics of DNA, the effect of the coupling strength of SWNT/DNA interface and the role of tube radius of nanotube contacts on the electronic transmission through the foregoing structure. Finally, a characteristic time was calculated for the electron transmission, which measures the delay caused by the tunnelling through the SWNT/DNA interface. The results clearly show that the calculated characteristic time and also the conductance of the system are sensitive to the coupling strength between DNA molecule and nanotube contacts.

Pressure Effects on the Ensemble Dynamics of Ubiquitin Inspected with Molecular Dynamics Simulations and Isotropic Reorientational Eigenmode Dynamics

According to NMR chemical shift data, the ensemble of ubiquitin is a mixture of “open” and “closed” conformations at rapid equilibrium. Pressure perturbations provide the means to study the transition between the two conformers by imposing an additional constraint on the system's partial molar volume. Here we use nanosecond-timescale molecular dynamics simulations to characterize the network of correlated motions accessible to the conformers at low- and high-pressure conditions. Using the isotropic reorientational eigenmode dynamics formalism to analyze our simulation trajectories, we reproduce NMR relaxation data without fitting any parameters of our model. Comparative analysis of our results suggests that the two conformations behave very differently. The dynamics of the “closed” conformation are almost unaffected by pressure and are dominated by large-amplitude correlated motions of residues 23–34 in the extended α-helix. The “open” conformation under conditions of normal pressure displays increased mobility, focused on the loop residues 17–20, 46–55, and 58–59 at the bottom of the core of the structure, as well as the C-terminal residues 69–76, that directly participate in key protein-protein interactions. For the same conformation, a pressure increase induces a loss of separability between molecular tumbling and internal dynamics, while motions between different backbone sites become uncorrelated.

Separation, detection, and quantification of hydroperoxides formed at side-chain and backbone sites on amino acids, peptides, and proteins

Hydroperoxides are major reaction products of radicals and singlet oxygen with amino acids, peptides, and proteins. However, there are few data on the distribution of hydroperoxides in biological samples and their sites of formation on peptides and proteins. In this study we show that normal-or reversed-phase gradient HPLC can be employed to separate hydroperoxides present in complex systems, with detection by postcolumn oxidation of ferrous xylenol orange to the ferric species and optical detection at 560 nm. The limit of detection (10–25 pmol) is comparable to chemiluminescence detection. This method has been used to separate and detect hydroperoxides, generated by hydroxyl radicals and singlet oxygen, on amino acids, peptides, proteins, plasma, and intact and lysed cells. In conjunction with EPR spin trapping and LC/MS/MS, we have obtained data on the sites of hydroperoxide formation. A unique fingerprint of hydroperoxides formed at α-carbon (backbone) positions has been identified; such backbone hydroperoxides are formed in significant yields only when the amino acid is part of a peptide or protein. Only side-chain hydroperoxides are detected with free amino acids. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants.

Reference energy extremal optimization: A stochastic search algorithm applied to computational protein design

We adapt a combinatorial optimization algorithm, extremal optimization (EO), for the search problem in computational protein design. This algorithm takes advantage of the knowledge of local energy information and systematically improves on the residues that have high local energies. Power-law probability distributions are used to select the backbone sites to be improved on and the rotamer choices to be changed to. We compare this method with simulated annealing (SA) and motivate and present an improved method, which we call reference energy extremal optimization (REEO). REEO uses reference energies to convert a problem with a structured local-energy profile to one with more random profile, and extremal optimization proves to be extremely efficient for the latter problem. We show in detail the large improvement we have achieved using REEO as compared to simulated annealing and discuss a number of other heuristics we have attempted to date.