Nuclear Overhauser Effect Experiments Research Articles

Enantioselective Relay Coupling of Perfluoroalkyl and Vinylogous Ketyl Radicals

Abstractβ‐Chiral carboxylic acids and their derivatives are highly valuable structural motifs in the fields of asymmetric synthesis and medicinal chemistry. However, the introduction of a sterically demanding sidechain to the β‐carbon, such as an all‐carbon quaternary center, remains a significant challenge in classical polar processes. Recently, N‐heterocyclic carbene (NHC) mediated coupling reactions involving persistent ketyl radicals have emerged as a promising strategy to assemble highly crowded carbon‐carbon bonds. Nevertheless, achieving enantioselectivity in these reactions remains highly challenging. In this work, we report our recent progress in controlling enantioselectivity for relay coupling of perfluoroalkyl and persistent vinylogous ketyl radicals. We developed a chiral bifunctional NHC‐squaramide catalyst that achieves high facial selectivity in a critical bond‐forming event involving the coupling of a congested tertiary carbon radical and vinylogous ketyl radical. Chiral carboxylates bearing an all‐carbon quaternary center at the β‐position can be prepared in good yield and excellent enantiomeric excess. Results from density functional theory (DFT) calculations and nuclear Overhauser effect (NOE) experiments indicate that the N,N’‐diaryl squaramide motif adopts an unusual syn‐syn conformation, enabling hydrogen bonding interactions with the enolate oxygen, thereby rigidifying the overall conformation of the transition state.

Enantioselective Relay Coupling of Perfluoroalkyl and Vinylogous Ketyl Radicals.

β-Chiral carboxylic acids and their derivatives are highly valuable structural motifs in the fields of asymmetric synthesis and medicinal chemistry. However, the introduction of a sterically demanding sidechain to the β-carbon, such as an all-carbon quaternary center, remains a significant challenge in classical polar processes. Recently, N-heterocyclic carbene (NHC) mediated coupling reactions involving persistent ketyl radicals have emerged as a promising strategy to assemble highly crowded carbon-carbon bonds. Nevertheless, achieving enantioselectivity in these reactions remains highly challenging. In this work, we report our recent progress in controlling enantioselectivity for relay coupling of perfluoroalkyl and persistent vinylogous ketyl radicals. We developed a chiral bifunctional NHC-squaramide catalyst that achieves high facial selectivity in a critical bond-forming event involving the coupling of a congested tertiary carbon radical and vinylogous ketyl radical. Chiral carboxylates bearing an all-carbon quaternary center at the β-position can be prepared in good yield and excellent enantiomeric excess. Results from density functional theory (DFT) calculations and nuclear Overhauser effect (NOE) experiments indicate that the N,N'-diaryl squaramide motif adopts an unusual syn-syn conformation, enabling hydrogen bonding interactions with the enolate oxygen, thereby rigidifying the overall conformation of the transition state.

Molecular Mechanisms Underlying Detection Sensitivity in Nanoparticle-Assisted NMR Chemosensing.

Nanoparticle-assisted nuclear magnetic resonance (NMR) chemosensing exploits monolayer-protected nanoparticles as supramolecular hosts to detect small molecules in complex mixtures via nuclear Overhauser effect experiments with detection limits down to the micromolar range. Still, the structure-sensitivity relationships at the basis of such detection limits are little understood. In this work, we integrate NMR spectroscopy and atomistic molecular dynamics simulations to examine the covariates that affect the sensitivity of different NMR chemosensing experiments [saturation transfer difference (STD), water STD, and high-power water-mediated STD]. Our results show that the intensity of the observed signals correlates with the number and duration of the spin-spin interactions between the analytes and the nanoparticles and/or between the analytes and the nanoparticles' solvation molecules. In turn, these parameters depend on the location and dynamics of each analyte inside the monolayer. This insight will eventually facilitate the tailoring of experimental and computational setups to the analyte's chemistry, making NMR chemosensing an even more effective technique in practical use.

E6AP AZUL interaction with UBQLN1/2 in cells, condensates, and an AlphaFold-NMR integrated structure

The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 biomolecular condensates invitro and E6AP interacts with UBQLN1/2 in cellulo. Steady-state and transfer nuclear Overhauser effect (NOE) experiments indicate direct interaction of AZUL with UBQLN1UBA. Intermolecular contacts identified by NOE spectroscopy (NOESY) data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We additionally identify an oligomerization domain directly adjacent to UBQLN1/2UBA (UBA adjacent [UBAA]) that is α-helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in condensates and cells.

Multifunctional Alternating “Bitter-Sweet” Macromolecular Architecture

The design and synthesis of a multifunctional macromolecular architecture featuring alternating cholic acid (CA) and glucose pendants in a polymer side-chain is reported. The target architecture was prepared by reversible addition–fragmentation chain-transfer copolymerization of styrene-conjugated CA (the bitter monomer) and acetyl-protected glucose appended maleimide (the sweet monomer) using the polyethylene glycol-conjugated chain transfer agent. Removal of the acetates resulted in amphiphilic “bitter-sweet” alternating copolymers that were self-assembled in aqueous media having CA containing bitter core and sugar-coated sweet shell. Dynamic light scattering measurements in water, field emission scanning electron microscopy, and transmission electron microscopy confirmed the formation of 40 to 75 nm sized micellar nanoscaffolds, depending on the chain-length of the copolymers. The nanoparticles successfully encapsulated hydrophobic molecules as witnessed via fluorescence spectroscopy using Nile red as an exemplary guest. Interestingly, the alternating copolymer recognized β-cyclodextrin (β-CD) through the formation of inclusion complexes with lateral cholate moieties in the polymer as evident from 2D NMR and nuclear Overhauser effect experiments. It is worth noting that the polymer and its inclusion complex were found to be capable of recognizing Concanavalin A (Con A), as shown by turbidimetric assay and isothermal titration calorimetry. Interestingly, the inclusion complex of the alternating copolymer showed significantly higher autofluorescence in the presence of Con A with respect to that of un-complexed one. Thus, the present study offers a simple way to prepare a multifunctional alternating copolymer having hydrophobic molecule encapsulation, inherent fluorescence, inclusion complex formation with β-CD, and lectin recognition capabilities.

Mechanistic Investigation of Drug Supersaturation in the Presence of Polysorbates as Solubilizing Additives by Solution Nuclear Magnetic Resonance Spectroscopy.

The introduction of solubilizing additives has historically been an attractive approach to address the ever-growing proportion of poorly water-soluble drug (PWSD) compounds within the modern drug discovery pipeline. Lipid-formulations, and more specifically micelle formulations, have garnered particular interest because of their simplicity, size, scalability, and avoidance of solid-state limitations. Although micelle formulations have been widely utilized, the molecular mechanism of drug solubilization in surfactant micelles is still poorly understood. In this study, a series of modern nuclear magnetic resonance (NMR) methods are utilized to gain a molecular-level understanding of intermolecular interactions and kinetics in a model system. This approach enabled the understanding of how a PWSD, 17β-Estradiol (E2), solubilizes within a nonionic micelle system composed of polysorbate 80 (PS80). Based on one-dimensional (1D) 1H chemical shift differences of E2 in PS80 solutions, as well as intermolecular correlations established from 1D selective nuclear Overhauser effect (NOE) and two-dimensional NOE spectroscopy experiments, E2 was found to accumulate within the palisade layer of PS80 micelles. A potential hydrogen-bonding interaction between a hydroxyl group of E2 and a carbonyl group of PS80 alkane chains may allow for stabilizing E2-PS80 mixed micelles. Diffusion and relaxation NMR analysis and particle size measurements using dynamic light scattering indicate a slight increase in the micellar size with increasing degrees of supersaturation, resulting in slower mobility of the drug molecule. Based on these structural findings, a theoretical orientation model of E2 molecules with PS80 molecules was developed and validated by computational docking simulations.

The Intermolecular NOE Depends on Isotope Selection: Short Range vs Long Range Behavior.

The nuclear Overhauser effect (NOE) is a powerful tool in molecular structure elucidation, combining the subtle chemical shift of NMR and three-dimensional information independent of chemical connectivity. Its usage for intermolecular studies, however, is fundamentally limited by an unspecific long-ranged interaction behavior. This joint experimental and computational work shows that proper selection of interacting isotopes can overcome these limitations: Isotopes with strongly differing gyromagnetic ratios give rise to short-ranged intermolecular NOEs. In this light, existing NOE experiments need to be re-evaluated and future ones can be designed accordingly. Thus, a new chapter on intermolecular structure elucidation is opened.

Non-uniform sampling for NOESY? A case study on spiramycin.

To date, most nuclear magnetic resonance (NMR)-based 3-D structure determinations of both small molecules and of biopolymers utilize the nuclear Overhauser effect (NOE) via NOESY spectra. The acquisition of high-quality NOESY spectra is a prerequisite for quantitative analysis providing accurate interatomic distances. As the acquisition of NOE build-ups is time-consuming, acceleration of the process by the use of non-uniform sampling (NUS) may seem beneficial; however, the quantitativity of NOESY spectra acquired with NUS has not yet been validated. Herein, NOESY spectra with various extents of NUS have been recorded, artificial NUS spectra with two different sampling schemes created, and by using two different NUS reconstruction algorithms the influence of NUS on the data quality was evaluated. Using statistical analyses, NUS is demonstrated to influence the accuracy of quantitative NOE experiments. The NOE-based distances show an increased error as the sampling density decreases. Weak NOE signals are affected more severely by NUS than more intense ones. The application of NUS with NOESY comes at two major costs: the interatomic distances are determined with lower accuracy and long-range correlations are lost.

Intra- and intermolecular H-bonding of benzotriazole UV stabilizers evidenced using 1D nuclear Overhauser effect experiments

The UV absorber protection mechanism of 2-hydroxyphenylbenzotriazoles is based upon energy dissipation via an excited state proton transfer from the phenolic OH group to the triazole nitrogen(s). Using 1H-NMR NOE experiments we have established that 2-(2’hydroxy-5′- methylphenyl)-benzotriazole (UVA1) exists in chloroform as an intramolecularly H-bonded form whereas in DMSO this bond is disrupted by the formation of intermolecular H-bonding to the solvent. Conversely, for compounds 2-(2′-hydroxy-3′,5′-di(1,1-dimethyl propane))-benzotriazole (UVA2), and 3′-methylene-hydantoin-2-(2′-hydroxy-5′-methylphenyl)-benzotriazole (UVA3) having bulky substituents ortho to the phenolic OH group 1H-NMR NOE experiments indicate that upon changing solvent from DMSO to chloroform the strength of the intramolecular H-bond is not appreciably affected. The implication of the H-bond strength upon the UV stabilizing effectiveness is discussed.

Synthesis of novel bis-sulfone derivatives and their inhibition properties on some metabolic enzymes including carbonic anhydrase, acetylcholinesterase, and butyrylcholinesterase.

In this study, a series of novel bis-sulfone compounds (2a-2j) were synthesized by oxidation of the bis-sulfides under mild reaction conditions. The bis-sulfone derivatives were characterized by 1 H-NMR, 13 C-NMR, Fourier-transform infrared spectroscopy, and elemental analysis techniques. Nuclear Overhauser effect experiments were performed to determine the orientation of the sulfonyl groups in bis-sulfone derivatives. Here, we report the synthesis and testing of novel bis-sulfone compound-based hybrid scaffold of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors for the development of novel molecules towardthe therapy of Alzheimer's disease. The novel synthesized bis-sulfone compounds demonstrated Ki values between 11.4 ± 3.4 and 70.7 ± 23.2 nM on human carbonic anhydrase I isozyme (hCA I), 28.7 ± 6.6 to 77.6 ± 5.6 nM on human carbonic anhydrase II isozyme (hCA II), 18.7 ± 2.61 to 95.4 ± 25.52 nM on AChE, and 9.5 ± 2.1 to 95.5 ± 1.2 nM on BChE enzymes. The results showed that novel bis-sulfone derivatives can have promising drug potential for glaucoma, leukemia, epilepsy, and Alzheimer's disease, which are associated with the high enzymatic activity of hCA I, hCA II, AChE, and BChE enzymes.

A Second Backbone: The Contribution of a Buried Asparagine Ladder to the Global and Local Stability of a Leucine-Rich Repeat Protein.

Parallel β-sheet-containing repeat proteins often display a structural motif in which conserved asparagines form a continuous ladder buried within the hydrophobic core. In such "asparagine ladders", the asparagine side-chain amides form a repetitive pattern of hydrogen bonds with neighboring main-chain NH and CO groups. Although asparagine ladders have been thought to be important for stability, there is little experimental evidence to support such speculation. Here we test the contribution of a minimal asparagine ladder from the leucine-rich repeat protein pp32 to stability and investigate lattice rigidity and hydrogen bond character using solution nuclear magnetic resonance (NMR) spectroscopy. Point substitutions of the two ladder asparagines of pp32 are strongly destabilizing and decrease the cooperativity of unfolding. The chemical shifts of the ladder side-chain HZ protons are shifted significantly downfield in the NMR spectrum and have low temperature coefficients, indicative of strong hydrogen bonding. In contrast, the HE protons are shifted upfield and have temperature coefficients close to zero, suggesting an asymmetry in hydrogen bond strength along the ladder. Ladder NH2 groups have weak 1H-15N cross-peak intensities; 1H-15N nuclear Overhauser effect and 15N CPMG experiments show this to be the result of high rigidity. Hydrogen exchange measurements demonstrate that the ladder NH2 groups exchange very slowly, with rates approaching the global exchange limit. Overall, these results show that the asparagine side chains are held in a very rigid, nondynamic structure, making a significant contribution to the overall stability. In this regard, buried asparagine ladders can be considered "second backbones" within the cores of their elongated β-sheet host proteins.

Structure determination protocol for transmembrane domain oligomers.

The transmembrane (TM) anchors of cell surface proteins have been one of the 'blind spots' in structural biology because they are generally very hydrophobic, sometimes dynamic, and thus difficult targets for structural characterization. A plethora of examples show these membrane anchors are not merely anchors but can multimerize specifically to activate signaling receptors on the cell surface or to stabilize envelope proteins in viruses. Through a series of studies of the TM domains (TMDs) of immune receptors and viral membrane proteins, we have established a robust protocol for determining atomic-resolution structures of TM oligomers by NMR in bicelles that closely mimic a lipid bilayer. Our protocol overcomes hurdles typically encountered by structural biology techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) when studying small TMDs. Here, we provide the details of the protocol, covering five major technical aspects: (i) a general method for producing isotopically labeled TM or membrane-proximal (MP) protein fragments that involves expression of the protein (which is fused to TrpLE) into inclusion bodies and releasing the target protein by cyanogen bromide (CNBr) cleavage; (ii) determination of the oligomeric state of TMDs in bicelles; (iii) detection of intermolecular contacts using nuclear Overhauser effect (NOE) experiments; (iv) structure determination; and (v) paramagnetic probe titration (PPT) to characterize the membrane partition of the TM oligomers. This protocol is broadly applicable for filling structural gaps of many type I/II membrane proteins. The procedures may take 3-6 months to complete, depending on the complexity and stability of the protein sample.

Transient NOE enhancement in solid-state MAS NMR of mobile systems

It has been known that the heteronuclear cross-relaxation affects the dilute S spin magnetization along the longitudinal direction, causing an overshoot phenomenon for those mobile systems in spin-lattice relaxation rate measurements. Here, we analyze the Solomon equations for an I-S system and derive the transient cross relaxation effect as to when an overshoot phenomenon would take place and what the maximum enhancement could be at the time of the overshoot. In order to utilize such a transient nuclear Overhauser effect (NOE), we first time apply it to dynamic solid samples by inverting the 1H magnetization prior to the excitation of the S spin. It is found that the overshoot depends on the ratio of the I and S spin-lattice relaxation rates, i.e. RSS/RII. When RSS/RII≫1, the maximum enhancement factor for transient NOE could be larger than that obtained in steady-state NOE experiments. Furthermore, transient NOE appears to be more efficient in terms of sensitivity enhancement of dilute spins in solid-state NMR of mobile systems than the traditional cross polarization scheme whose efficiency is greatly compromised by molecular mobility. A sample of natural abundance l-isoleucine amino acid, in which the spin-lattice relaxation rates for the four methyl carbons are different, has been used to demonstrate sensitivity enhancement factors under various experimental schemes.

Synthesis and spectral evaluation of 5,10,15,20-tetrakis(3,4-dibenzyloxyphenyl)porphyrin

Abstract Porphyrins are of interest in many applications that involve electron transfer and absorption of light, such as solar energy and photodynamic cancer therapy. The newly synthesized 5,10,15,20-tetrakis(3,4-dibenzyloxyphenyl)porphyrin, TDBOPP, was characterized using 1H NMR, 13C NMR, UV-vis and fluorescence spectroscopy and MALDI-TOF/TOF high resolution mass spectrometry. Standard 1H NMR and 13C NMR experiments coupled with nuclear Overhauser effect (NOE) experiments confirmed the structure of the compound. The expected M+ and [M+H]+ ions are observed in the MALDI-TOF/TOF mass spectrum. The UV-vis absorption spectrum of TDBOPP shows a Soret band at 424 nm and three Q bands at 519 nm, 556 nm, and 650 nm with molar absorptivity 3.6×105 cm−1 m −1, 1.6×104 cm−1 m −1, 1.0×104 cm−1 m −1 and 5.3×103 cm−1 m −1, respectively. Excitation at 424 nm gives emission at 650 nm. The quantum yield of the porphyrin is 0.11.

Analysis of six ‘neuro‐enhancing’ phenidate analogs

Six collected phenidates, i.e., 4-methylmethylphenidate, 3,4-dichloromethylphenidate, ethylphenidate, 3,4-dichloroethylphenidate, ethylnaphthidate, and N-benzyl-ethylphenidate were fully characterized by means of X-ray, nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), electrospray ionization-tandem mass spectrometry (ESI-MS/MS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GC solid-state IR analysis. Crystallography revealed the exclusive presence of the threo-configuration. Steric crowding induced by N-benzyl substitution at the piperidine moiety prompted an adoption of an unexpected axial positioning of substituents on the piperidine moiety in the crystal state as opposed to the exclusive equatorial positioning encountered in N-unsubstituted phenidate analogues. Gas phase computations of the relative lowest energy conformers confirm that the axial positioning appears to be favoured over the equatorial positioning; in solution, however, equatorial positioning is predominant according to nuclear Overhauser effect experiments. All samples, mainly originating from China, had a good to very good degree of purity indicative of their professional chemical synthesis. Routine analysis of these drugs by GC-MS revealed thermal decomposition of phenidate analogues in the injection port and/or on column to 2-aryl-ethyl-acetates and 2,3,4,5-tetrahydropyridines. The decomposition pathway was suggested to proceed via a 6-membered transition state which was supported by density functional theory (DFT) computations. Fragmentation pathways of decomposition products as well as the corresponding electron ionization (EI) mass spectra are provided. The thermal instability might thus render smoking or 'vaping' of these drugs a less effective route of administration. The analytical fingerprints of six structurally diverse phenidate analogues provide a helpful reference to forensic chemists in charge of identifying new psychoactive substances. Copyright © 2017 John Wiley & Sons, Ltd.

Efficient Approaches for the Synthesis of Substituted Thiazolo[3,2‐a]‐benzimidazole‐Phosphonates and ‐Phosphinic Diamide Derivatives

AbstractPhosphrylation of E‐3‐(dimethylamino)‐1‐(3‐methylbenzo[d]‐thiazolo[3,2‐a]imidazol‐2‐yl)‐prop‐2‐en‐1‐one (1) and 1‐(3‐methylbenzo[d]thiazolo[3,2‐a]imidazol‐2‐yl)ethanone (12) was studied in detail. The enaminone (1) reacted with trialkyl phosphites, dialkylphosphonates, and tris(dialkylamino)‐phosphines to afford the corresponding (E/Z) thiazolobenzimidazol‐3‐oxoprop‐1‐enyl‐phosphonate and phosphonic diamides. The reactions proceeded via phospha‐Michael (P‐Michael) addition reaction, followed by extrusion of HNMe2 moiety and intramolecular migration of the phosphono‐alkyl group. Only the major E‐isomer of the respective oxopropenyl phosphonates was isolated by fractional crystallization (∼ 65% yield) in a pure form whereas the two isomers were chromatography separated and identified in the second case of the respective phosphonic diamide mixtures. The trans‐configuration is also confirmed by nuclear Overhauser effect (NOE) experiments. On the other hand, substituted thiazolobenzimidazole‐α‐hydroxyphosphonate‐ and α‐hydroxyphosphinic diamide derivatives were produced from the reaction of the 2‐acetyl thiazolo[3,2‐a]benzimidazole (12) with the same P(III) reagents via Abramov 1, 2‐addition reaction.

Extraction of distance restraints from pure shift NOE experiments

NMR techniques incorporating pure shift methods to improve signal resolution have recently attracted much attention, owing to their potential use in studies of increasingly complex molecular systems. Extraction of frequencies from these simplified spectra enables easier structure determination, but only a few of the methods presented provide structural parameters derived from signal integral measurements. In particular, for quantification of the nuclear Overhauser effect (NOE) it is highly desirable to utilize pure shift techniques where signal overlap normally prevents accurate signal integration, to enable measurement of a larger number of interatomic distances. However, robust methods for the measurement of interatomic distances using the recently developed pure shift techniques have not been reported to date.In this work we discuss some of the factors determining the accuracy of measurements of signal integrals in interferogram-based Zangger-Sterk (ZS) pure shift NMR experiments. The ZS broadband homodecoupling technique is used in different experiments designed for quantitative NOE determination from pure shift spectra. It is shown that the techniques studied can be used for quantitative extraction of NOE-derived distance restraints, as exemplified for the test case of strychnine.

Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase.

Protein-ligand interaction is often characterized using polarization transfer by the intermolecular nuclear Overhauser effect (NOE). For such NOE experiments, hyperpolarization of nuclear spins presents the opportunity to increase the spin magnetization, which is transferred, by several orders of magnitude. Here, folic acid, a ligand of dihydrofolate reductase (DHFR), was hyperpolarized on (1)H spins using dissolution dynamic nuclear polarization (D-DNP). Mixing hyperpolarized ligand with protein resulted in observable increases in protein (1)H signal predominantly in the methyl group region of the spectra. Using (13)C single quantum selection in a series of one-dimensional spectra, the carbon chemical shift ranges of the corresponding methyl groups can be elucidated. Signals observed in these hyperpolarized spectra could be confirmed using 3D isotope filtered NOESY spectra, although the hyperpolarized spectra were obtained in single scans. By further correlating the signal intensities observed in the D-DNP experiments with the occurrence of short distances in the crystal structure of the protein-ligand complex, the observed methyl proton signals could be matched to the chemical shifts of six amino acids in the active site of DHFR-folic acid binary complex. These data demonstrate that (13)C chemical shift selection of protein resonances, combined with the intrinsic selectivity towards magnetization originating from the initially hyperpolarized spins, can be used for site specific characterization of protein-ligand interactions.

CASP11--An Evaluation of a Modular BCL::Fold-Based Protein Structure Prediction Pipeline.

In silico prediction of a protein’s tertiary structure remains an unsolved problem. The community-wide Critical Assessment of Protein Structure Prediction (CASP) experiment provides a double-blind study to evaluate improvements in protein structure prediction algorithms. We developed a protein structure prediction pipeline employing a three-stage approach, consisting of low-resolution topology search, high-resolution refinement, and molecular dynamics simulation to predict the tertiary structure of proteins from the primary structure alone or including distance restraints either from predicted residue-residue contacts, nuclear magnetic resonance (NMR) nuclear overhauser effect (NOE) experiments, or mass spectroscopy (MS) cross-linking (XL) data. The protein structure prediction pipeline was evaluated in the CASP11 experiment on twenty regular protein targets as well as thirty-three ‘assisted’ protein targets, which also had distance restraints available. Although the low-resolution topology search module was able to sample models with a global distance test total score (GDT_TS) value greater than 30% for twelve out of twenty proteins, frequently it was not possible to select the most accurate models for refinement, resulting in a general decay of model quality over the course of the prediction pipeline. In this study, we provide a detailed overall analysis, study one target protein in more detail as it travels through the protein structure prediction pipeline, and evaluate the impact of limited experimental data.

Lipopolysaccharide-bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy.

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad-spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram-negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram-negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α-helical structure in a solution containing LPS. For NMR experiments, we expressed (15) N-labeled and (13) C-labeled CP1 in bacterial cells and successfully assigned almost all backbone and side-chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr-NOE) experiments in LPS. We performed (15) N-edited and (13) C-edited Tr-NOE spectroscopy for CP1 bound to LPS. Tr-NOE peaks were observed at the only C-terminal region of CP1 in LPS. The results of structure calculation indicated that the C-terminal region (Lys15-Gly29) formed the well-defined α-helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A.