Solution-state Structure Research Articles

Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM

Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand breaks (DSBs) forms DNA-PK holoenzyme as a critical initiating step for non-homologous end joining (NHEJ) repair of DSBs produced by radiation and chemotherapies. Advanced cryo-electron microscopy (cryo-EM) imaging together with breakthrough macromolecular X-ray crystal (MX) structures of KU and DNA-PKcs recently enabled visualization of the ∼600 kDa DNA-PK assembly at near atomic resolution. These important static structures provide the foundation for definition and interpretation of functional movements crucial to mechanistic understanding that can be tested through solution state structure analysis. We herein therefore leverage Cryo-EM and MX structures for the interpretation of synchrotron small-angle X-ray scattering (SAXS) data on DNA-PK conformations in solution to inform the structural mechanism for NHEJ initiation. SAXS, which measures thermodynamic solution-state conformational states and assemblies outside of cryo- and solid-state conditions, unveils the inherent flexibility of KU, DNA-PKcs and DNA-PK. The combined structural measurements reveal mobility of KU80 C-terminal region (KU80CTR), motion/plasticity of HEAT (DNA-PKcs Huntingtin, Elongation Factor 3, PP2 A, and TOR1) regions, allosteric switching upon DNA-PKcs autophosphorylation, and dimeric arrangements of DNA-PK assembly. Importantly, the results uncover displacement of the N-terminal HEAT domain during autophosphorylation as suitable for a regulated release mechanism of DNA-PKcs from DNA-PK to control unproductive access to toxic and mutagenic DNA repair intermediates. These integrated analyses show that the marriage of SAXS with cryo-EM leverages the strengths of both techniques to enable assessment of functional conformations and flexibility defining atomic-resolution molecular mechanisms for DSB repair.

Gold(I), silver(I) and copper(I) complexes of 2,4,6-trimethylphenyl-3-benzoylthiourea; synthesis and biological applications

2,4,6-Trimethylphenyl-3-benzoylthiourea (L) and its gold(I), silver(I) and copper(I) complexes were synthesized. Treatment of L with HAuCl4, AgNO3 and CuI in CH3CN afforded complexes of general representation, M(L)X, where M/X = Au/Cl (1), Ag/NO3 (2), Cu/I (3). Reaction with Au3+ salt afforded two products, wherein complex containing Au+ (reduced under normal and aerobic reaction conditions ca. 66% on NMR time scale) was confirmed by X-ray diffraction for a single crystal of complex 1. The minor fraction (ca. 33%, from 1H NMR data) in the reaction mixture was identified as complex 4 with a general formula [AuL2]Cl3. Reaction with AgNO3 and CuI exclusively afforded pure products 2 and 3. Ligand L and complexes 1–4 were characterized by UV/visible, FT-IR and 1H-, 13C NMR spectroscopy. Complex 1 is crystalline in nature and good quality crystals were obtained which allowed for collection of diffraction data and the structure was thus confirmed. Solution state and solid state structures have been compared. The resultant complexes were tested for their cytotoxicity, antibacterial, antifungal, antioxidant and DNA binding activities. Complex 1 (Au based) showed promising activities in comparison with the available selected drugs used as standards.

Structural and mechanistic insights into Hsp104 function revealed by synchrotron X-ray footprinting

Hsp104 is a hexameric AAA+ ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5'-O-(thiotriphosphate)). Comparing side-chain solvent accessibilities between these three states illuminated aspects of Hsp104 structure and guided design of Hsp104 variants to probe the disaggregase mechanism in vitro and in vivo We established that Hsp104 hexamers switch from a more-solvated state in ADP to a less-solvated state in ATPγS, consistent with switching from an open spiral to a closed ring visualized by cryo-EM. We pinpointed critical N-terminal domain (NTD), NTD-nucleotide-binding domain 1 (NBD1) linker, NBD1, and middle domain (MD) residues that enable intrinsic disaggregase activity and Hsp70 collaboration. We uncovered NTD residues in the loop between helices A1 and A2 that can be substituted to enhance disaggregase activity. We elucidated a novel potentiated Hsp104 MD variant, Hsp104-RYD, which suppresses α-synuclein, fused in sarcoma (FUS), and TDP-43 toxicity. We disambiguated a secondary pore-loop in NBD1, which collaborates with the NTD and NBD1 tyrosine-bearing pore-loop to drive protein disaggregation. Finally, we defined Leu-601 in NBD2 as crucial for Hsp104 hexamerization. Collectively, our findings unveil new facets of Hsp104 structure and mechanism. They also connect regions undergoing large changes in solvation to functionality, which could have profound implications for protein engineering.

Effect of Heterocyclic Ring on LnIII Coordination, Luminescence and Extraction of Diamides of 2,2'-Bipyridyl-6,6'-Dicarboxylic Acid.

We have synthesized and examined several complexes of lanthanides with diamides of 2,2′-bipyridyl-6,6′-dicarboxylic acid bearing various hetaryl-based side chains for the elucidation of the effect of the heterocycle on the structure and properties of the ligands. The multigram scale methods for the preparation of various N-alkyl-hetaryls and their diamides were developed. The solid state structure of 6-methyl-2-pyridylamide of 2,2′-bipyridyl-6,6′-dicarboxylic acid possesses a flat structure where the conformation is completely different from that previously observed for N-alkylated 2,2′-bipyridyl-6,6′-dicarboxamides and 2,6-pyridinedicarboxamides. The complexes of new ligands were synthesized and NMR and X-Ray studied their structure in solution and solid state. The results demonstrate that complexes possess the same structures both in solid state and in solution. Stability constants of the complexes were less when comparing with dimethyl-substituted diamides, but higher than for unsubstituted dianilide. Contrarily, the extraction ability of 2-pyridyl-diamide is significantly lower than for corresponding anilide. Specific interaction of extractant with solvent molecules, which is not available for electron-sink pyridine amides, can explain this. The luminescence of new Eu complexes was significantly higher than for all previously 2,2′-bipyridyl-6,6′-dicarboxamides and QY reaches 18%. Asymmetry ratios of Eu complexes were 25% higher when compared other complexes with 2,2′-bipyridyl-6,6′-dicarboxamides, which indicates large deviation from the inversion center.

Effect of Solvents on the Solution State and Film Condensed State Structures of a Polyfluorene Conjugated Polymer in the Dynamic Evolution Process from Solution to Film

Although much progress has been made in basic research on conjugated polymers and their practical applications in optoelectronic devices, so far research on the solution state, especially on the effect of solvent on conjugated polymers solution state and films condensed state structure in the dynamic evolution process from solution to film, is scarce. In this research, we focused on a special polyfluorene conjugated polymer, poly(9-(4-(octyloxy)phenyl)-2,7-fluoren-9-ol) (PPFOH), hydrogen bonding with supramolecular conjugated polymers in which intermolecular hydrogen bonds could be formed with different solvents, to explore the effect of solvents on PPFOH’s solution state and the film’s condensed state structure in the dynamic evolution process from solution to film. Dynamic/static light scattering, giving shape parameters and fractal dimension, UV–vis absorption spectra, photoluminescence spectra, fluorescence lifetime measurements, and transmission electron microscopy were used for this research. It was...

Polyglutamine Solution-State Structural Propensity Is Repeat Length Dependent.

Expanded polyglutamine (polyQ) tracts in proteins, which are known to induce their aggregation, are associated with numerous neurodegenerative diseases. Longer polyQ tracts correlate with faster protein aggregation kinetics and a decreased age of onset for polyQ disease symptoms. Here, we use UV resonance Raman spectroscopy, circular dichroism spectroscopy, and metadynamics simulations to investigate the solution-state structures of the D2Q15K2 (Q15) and D2Q20K2 (Q20) peptides. Using metadynamics, we explore the conformational energy landscapes of Q15 and Q20 and investigate the relative energies and activation barriers between these low-energy structures. We compare the solution-state structures of D2Q10K2 (Q10), Q15, and Q20 to determine the dependence of polyQ structure on the Q tract length. We show that these peptides can adopt two distinct monomeric conformations: an aggregation-resistant PPII-like conformation and an aggregation-prone β-strand-like conformation. We find that longer polyQ peptides have an increased preference for the aggregation-prone β-strand-like conformation. This preference may play an important role in the increased aggregation rate of longer polyQ peptides that is thought to lead to decreased neurodegenerative disease age of onset for polyQ disease patients.

Development of Synchrotron Footprinting at NSLS and NSLS-II.

First developed in the 1990's at the National Synchrotron Light Source, xray synchrotron footprinting is an ideal technique for the analysis of solution-state structure and dynamics of macromolecules. Hydroxyl radicals generated in aqueous samples by intense x-ray beams serve as fine probes of solvent accessibility, rapidly and irreversibly reacting with solvent exposed residues to provide a "snapshot" of the sample state at the time of exposure. Over the last few decades, improvements in instrumentation to expand the technology have continuously pushed the boundaries of biological systems that can be studied using the technique. Dedicated synchrotron beamlines provide important resources for examining fundamental biological mechanisms of folding, ligand binding, catalysis, transcription, translation, and macromolecular assembly. The legacy of synchrotron footprinting at NSLS has led to significant improvement in our understanding of many biological systems, from identifying key structural components in enzymes and transporters to in vivo studies of ribosome assembly. This work continues at the XFP (17-BM) beamline at NSLS-II and facilities at ALS, which are currently accepting proposals for use.

Structural and functional characterisation of the entry point to pyocyanin biosynthesis in Pseudomonas aeruginosa defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass.

In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.

Exploration of the Solid- and Solution-State Structures and Electrochemical Properties of CeIV(atrane) Complexes.

Chemical oxidation of cerium complexes can be unpredictable because of labile metal-ligand bonds leading to ligand redistribution. The use of tripodal frameworks such as silyl-substituted tren ligands (NN'3 = [N(CH2CH2N(SiMe2tBu))3]3-) and a tris(hydroxylaminato) ligand, [((2- tBuNO)C6H4CH2)3N]3- (TriNOx3-), has been shown to mitigate ligand redistribution effects to allow access to tetravalent cerium complexes with different apical ligands. In the current work, the coordination chemistry of CeIV with the related tripodal atrane3- (H3atrane = [N(CH2C(CH3)2OH)3]) ligand framework was examined. A series of CeIV(atrane) complexes with supporting chloride, silylamide, and aryloxide ligands were synthesized and characterized by X-ray crystallography. The solution-state behaviors of these complexes were studied using 1H and diffusion-ordered (DOSY) NMR spectroscopies. The electrochemical stabilization of the CeIV cation within the atrane3- framework was examined. Our results showed that a combination of suitable apical ligands and the atrane framework provided excellent stabilization for the CeIV cation.

Synthesis, structures, and investigation of noncovalent interactions of 1,3-dimethyl-5-(4ʹ/3ʹ-pyridylazo)-6-aminouracil and their Ni(II) complexes

In this research paper we report synthesis of two planar ligands, namely 1,3-dimethyl-5-(4ʹ/3ʹ-pyridylazo)-6-aminouracil: HL1 (1) for 4ʹ-pyridyazo- and HL2 (2) for 3ʹ-pyridyazo-group and, moreover, their nickel (II) square planar complexes ([NiII(L1)2] (3) and ([NiII(L2)2] (4)). The report is also included solid and solution state structure of the compounds and study of noncovalent stacking interactions. Crystal structures of the ligands, 1 and 2 are monoclinic with alike space group P21/c and the complex 3 is also monoclinic with space group C2/c. The molecular electrostatic potential (MEP) analysis shows that the ligands possess both π-basic and π-acid character in the same framework over the center of the pyridine and the pyrimidine rings, respectively, favoring electrostatically enhanced π-π stacking interaction among the molecular units. Upon complexation, π-acidity of the pyrimidine ring increases, and the complex 3 exhibits lone pair (lp)-π stacking interaction involving lone pair of the pyridine N-atom and π-acid pyrimidine plane. The study evidences that the ligands exist in azo-enamine-keto tautomer in solid and hydrazone-imine-keto in solution state and get coordinated with nickel (II) via azo-N and uracil-6-NH, to form a distorted square planar complex. Electronic absorption bands/energies obtained from experimental and theoretical (TDDFT) study are indeed alike, giving reliability to the level of theory and the absorption band assignments. Moreover, the synthesized compounds containing uracil and pyridine fragments exhibit π-π/lp-π interactions with considerable energy for stabilization of their assemblies in the solid state.

Conformational Sampling of the Intrinsically Disordered C-Terminal Tail of DERA Is Important for Enzyme Catalysis.

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes the reversible conversion of acetaldehyde and glyceraldehyde-3-phosphate into deoxyribose-5-phosphate. DERA is used as a biocatalyst for the synthesis of drugs such as statins and is a promising pharmaceutical target due to its involvement in nucleotide catabolism. Despite previous biochemical studies suggesting the catalytic importance of the C-terminal tyrosine residue found in several bacterial DERAs, the structural and functional basis of its participation in catalysis remains elusive because the electron density for the last eight to nine residues (i.e., the C-terminal tail) is absent in all available crystal structures. Using a combination of NMR spectroscopy and molecular dynamics simulations, we conclusively show that the rarely studied C-terminal tail of E. coli DERA (ecDERA) is intrinsically disordered and exists in equilibrium between open and catalytically relevant closed states, where the C-terminal tyrosine (Y259) enters the active site. Nuclear Overhauser effect distance restraints, obtained due to the presence of a substantial closed state population, were used to derive the solution-state structure of the ecDERA closed state. Real-time NMR hydrogen/deuterium exchange experiments reveal that Y259 is required for efficiency of the proton abstraction step of the catalytic reaction. Phosphate titration experiments show that, in addition to the phosphate-binding residues located near the active site, as observed in the available crystal structures, ecDERA contains previously unknown auxiliary phosphate-binding residues on the C-terminal tail which could facilitate in orienting Y259 in an optimal position for catalysis. Thus, we present significant insights into the structural and mechanistic importance of the ecDERA C-terminal tail and illustrate the role of conformational sampling in enzyme catalysis.

The Curious Case of the OZ439 Mesylate Salt: An Amphiphilic Antimalarial Drug with Diverse Solution and Solid State Structures.

Efforts to develop orally administered drugs tend to place an exceptional focus on aqueous solubility as this is an essential criterion for their absorption in the gastrointestinal tract. In this work we examine the solid state behavior and solubility of OZ439, a promising single-dose cure for malaria being developed as the highly water-soluble mesylate salt. The aqueous phase behavior of the OZ439 mesylate salt was determined using a combination of small angle neutron and X-ray scattering (SANS and SAXS, respectively). It was found that this salt has low solubility at low concentrations with the drug largely precipitated in free base aggregates. However, with increasing concentration these crystalline aggregates were observed to dissociate into cationic micelles and lamellar phases, effectively increasing the dissolved drug concentration. It was also found that the dissolved OZ439 spontaneously precipitated in the presence of biologically relevant anions, which we attribute to the high lattice energies of most of the salt forms of the drug. These findings show that aqueous solubility is not always what it seems in the context of amphiphilic drug molecules and highlights that its use as the principal metric in selecting drug candidates for development can be perilous.

Solution-state structures of the cellulose model pullulan in lithium chloride/N,N-dimethylacetamide

Pullulan is soluble in both N,N-dimethylacetamide (DMAc) and LiCl/DMAc. Here, the solution-state structures of pullulan in these two solvents were studied by size-exclusion chromatography with multi-angle laser light scattering (SEC/MALLS) and nuclear magnetic resonance (NMR) analyses. SEC/MALLS and off-line refractive index analyses revealed that the hydroxy groups of pullulan interact with LiCl in DMAc, as observed previously for cellulose. The nine different COH protons of pullulan in DMAc and LiCl/DMAc appeared independently in NMR spectra and were successfully assigned. When LiCl was present, the degree of dissociation of all COH protons of pullulan was increased by Li+ and Cl− ions, and the conformations of the COH groups became stable and restricted in DMAc. Based on the obtained results, the solution-state structures of cellulose in LiCl/DMAc are hypothesized to contain similar interactions between the cellulose hydroxy groups, and Li+ and Cl− in LiCl/DMAc to those observed for pullulan.

Unraveling the Solution-State Supramolecular Structures of Donor-Acceptor Polymers and their Influence on Solid-State Morphology and Charge-Transport Properties.

Polymer self-assembly in solution prior to film fabrication makes solution-state structures critical for their solid-state packing and optoelectronic properties. However, unraveling the solution-state supramolecular structures is challenging, not to mention establishing a clear relationship between the solution-state structure and the charge-transport properties in field-effect transistors. Here, for the first time, it is revealed that the thin-film morphology of a conjugated polymer inherits the features of its solution-state supramolecular structures. A "solution-state supramolecular structure control" strategy is proposed to increase the electron mobility of a benzodifurandione-based oligo(p-phenylene vinylene) (BDOPV)-based polymer. It is shown that the solution-state structures of the BDOPV-based conjugated polymer can be tuned such that it forms a 1D rod-like structure in good solvent and a 2D lamellar structure in poor solvent. By tuning the solution-state structure, films with high crystallinity and good interdomain connectivity are obtained. The electron mobility significantly increases from the original value of 1.8 to 3.2 cm2 V-1 s-1 . This work demonstrates that "solution-state supramolecular structure" control is critical for understanding and optimization of the thin-film morphology and charge-transport properties of conjugated polymers.

Mechanism of Discrimination of 8-Oxoguanosine versus Guanosine by Escherichia coli Fpg.

The mutagenic 8-oxoguanosine monophosphate, the predominant product of DNA oxidation, is excised by formamidopyrimidine glycosylase (Fpg) in bacteria. The mechanism of recognition of 8-oxodG, which differs subtly from its normal counterpart, guanosine monophosphate (dG), by Escherichia coli Fpg remains elusive due to the lack of structural data of E. coli Fpg bound to 8-oxodG. Here, we present solution-state structure of 8-oxodG oligomer bound to E. coli E3Q Fpg using UV resonance Raman (UVRR) spectroscopy. The vibrational spectra report on the π-stacking and hydrogen bonding interactions established by 8-oxodG with E. coli E3Q Fpg. Furthermore, we report on the interactions of E. coli E3Q Fpg with the normal, undamaged nucleotide, dG. We show that E. coli Fpg recognizes 8-oxodG and dG through their C2-amino group but only 8-oxodG forms extensive contacts with E. coli Fpg. Our findings provide a basis for mechanism of lesion recognition by E. coli Fpg.

SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline

Synchrotron X-ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small-angle X-ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X-ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X-ray footprinting of biomolecules performed for the first time at the X-ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped-flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X-ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid-associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high-resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent-exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X-ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X-ray footprinting of biomolecules.

Chemical shift assignments of Ribosomal protein S1 from Mycobacterium tuberculosis.

RpsA, also known as ribosomal protein S1, is an essential protein required for translation initiation of mRNAs when their Shine-Dalgarno sequence is degenerated (Sorensen et al. 1998). In addition, RpsA of Mycobacterium tuberculosis (M. tb) is involved in trans-translation, which is an effective system mediated by tmRNA-SmpB to release stalled ribosomes from mRNA in the presence of rare codons (Keiler 2008). Shi et al. found that POA binds to RpsA of Mtb and disrupts the formation of RpsA-tmRNA complex (Shi et al. 2011) and mutations at the C-terminus of RpsA confer PZA resistance. The previous work reported the pyrazinoic acid-binding domain of RpsA (Yang et al. Mol Microbiol 95:791-803, 2015). However, the HSQC spectra of the isolated S1 domain does not overlap with that of MtRpsA280-438, suggesting that substantial interactions occur between the flexible C-terminus and the S1 domain in MtRpsA .To further study the PZA resistance and how substantial interactions influence/affect protein structure, using heteronuclear NMR spectroscopy, we have completed backbone and side-chain 1H, 15N, 13C chemical shift assignments of MtRpsA280-438 which contains S1 domain and the flexible C-terminus. These NMR resonance assignments provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.

Solution and solid state structures and magnetism of a series of linear trinuclear compounds with a hexacoordinate LnIII and two terminal NiII centers.

Reported are the syntheses, structures and magnetic properties, also by NMR spectroscopy in solution, of a series of 13 linear trinuclear 3d-4f compounds with a lanthanide(iii) surrounded by two NiII ions, NiLnIII, where the central LnIII is hexacoordinate. For three of the crystal structures, an additional H2O molecule is coordinated to the central LnIII ion, leading to a monocapped trigonal prismatic structure. However, NMR spectroscopy indicates that in solution, these complexes also have a hexacoordinate LnIII center. The solution magnetic anisotropies, determined by NMR spectroscopy, indicate that the axial components of the anisotropies are relatively small and that the DyIII derivative might therefore not exhibit single molecule magnetism. The axial anisotropies determined by NMR spectroscopy are in good agreement with the expectations based on the distorted trigonal prismatic ligand field.

Spectrophotometric Determination of Tin(II) with 6-Chloro-2-(2'-furyl)-3-Hydroxy-7-Methyl-4-Oxo-4H-1-Benzopyran and Its Solution State Structure Study by DFT

Spectrophotometric Determination of Tin(II) with 6-Chloro-2-(2'-furyl)-3-Hydroxy-7-Methyl-4-Oxo-4<i>H</i>-1-Benzopyran and Its Solution State Structure Study by DFT

Solution State Structure of P1, the Mimetic Peptide Derived from IgM Antigen Apo B-100 by NMR

Apolipoprotein B-100 (Apo-B100) is a major component of low density lipoprotein (LDL). Apo B-100 protein has 4,536 amino acid sequence and these amino acids are classified into peptide groups A to G with subsequent 20 amino acids (P1-P302). The peptide groups were act as immunoglobulin (Ig) antigens which oxidized via malondialdehyde (MDA). The mimetic peptide P1 (EEEMLENVSLVCPKDAT RFK) out of D-group peptides carrying the highest value of IgG antigens were selected for structural studies that may provide antigen specificity. Circular Dichroism (CD) spectra were measured for peptide secondary structure in the range of 190-250 nm. Experimental results show that P1 exhibit partial of <TEX>${\beta}-sheet$</TEX> and random coil structure. Homonuclear (COSY, TOCSY, NOESY) 2D-NMR experiments were carried out for NMR signal assignments and structure determination for P1. On the basis of these completely assigned NMR spectra and distance data, distance geometry (DG) and Molecular dynamics (MD) were carried out to determine the structures of P1. The proposed structure was selected by comparisons between experimental NOE spectra and back calculated 2D NOE results from determined structure showing acceptable agreement. The total Root-Mean-Square-Deviation (RMSD) value of P1 obtained upon superposition of all atoms was in the range <TEX>$0.33{\AA}$</TEX>. The solution state P1 has mixed structure of <TEX>${\beta}-sheet$</TEX> (Glu[1] to Cys[12]) and random coil (Pro[13] to Lys[20]). These NMR results are well consistent with secondary structure from experimental results of circular dichroism. Structural studies based on NMR may contribute to the studies of atherosclerosis and observed conformational characteristics of apo B-100 in LDL using monoclonal antibodies.