NMR Structures (nmr + structure)

Distribution by Scientific Domains
Distribution within Chemistry

Terms modified by NMR Structures

  • nmr structure determination

  • Selected Abstracts

    Cover Picture: A Cyclic CCK8 Analogue Selective for the Cholecystokinin Type A Receptor: Design, Synthesis, NMR Structure and Binding Measurements (ChemBioChem 11/2003)

    CHEMBIOCHEM, Issue 11 2003
    Stefania De Luca Dr.
    Abstract The cover picture shows a molecular model of the interaction between the new CCK8 analogue, Cycle29,34[Dpr29,Lys34]-CCK8 (shown as a CPK model) and the receptor fragment CCKA -R(1,47) (represented by a pink ribbon). The introduction of the cyclic constraint between the Dpr29 sidechain and the CCK8 C terminus (Lys34) decreases the flexibility of the molecule to stabilize the bioactive conformation of Cycle29,34[Dpr29,Lys34]-CCK8. The Trp30 and Met31 side chains are in favorable orientations for interaction with the CCKA receptor. Expansions of the aromatic/amide regions of the 1H NMR spectra of Cycle29,34[Dpr29,Lys34]-CCK8 in aqueous solution (top) and in presence of dodecylphosphocholine-d38 micelles (bottom) are shown in the inset. Further details can be found in the article by Morelli and co-workers on p. 1176 ff. [source]

    Cover Picture: NMR Structure of the Single QALGGH Zinc Finger Domain from the Arabidopsis thaliana SUPERMAN Protein (ChemBioChem 2-3/2003)

    CHEMBIOCHEM, Issue 2-3 2003
    Carla Isernia Prof.
    Abstract The cover picture shows the NMR structure of the SUPERMAN zinc finger domain, which is the first high-resolution structure of a classical zinc finger domain from a plant protein. The structure consists of a very well-defined ,,, motif, typical of all the other Cys2,His2 zinc fingers so far structurally characterized. As a consequence, the QALGGH sequence, which is highly conserved in plant protein classical zinc finger domains, is located at the N terminus of the , helix. Interestingly, this domain region, in animal protein zinc fingers, is constituted of hypervariable residues deputed to the recognition of the DNA bases. Therefore, a peculiar DNA recognition code for the QALGGH zinc finger domain is proposed in the article by Fattorusso and co-workers on p. 171 ff. [source]

    ,-Conotoxin CVIB differentially inhibits native and recombinant N- and P/Q-type calcium channels

    Leonid Motin
    Abstract ,-Conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist ,-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. ,-Conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 0.05 and 7.64 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (, 66% of total) appeared to be irreversible for >,30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (, 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (, 22% of total) was attributable to P/Q-type VGCCs. ,-Conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (CaV2.2) and P/Q- (CaV2.1), but not R- (CaV2.3) type VGCCs expressed in Xenopus oocytes. The ,2,1 auxiliary subunit co-expressed with CaV2.2 and CaV2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. ,-Conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information. [source]

    Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase

    FEBS JOURNAL, Issue 21 2006
    Xun-Cheng Su
    DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1,171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1,171 of the E. coli DnaB helicase with significant affinity. [source]

    Modeling zinc sulfhydryl bonds in zinc fingers

    Johan Bredenberg
    Abstract Molecular dynamics simulations have been carried out employing three different model descriptions of the zinc sulfhydryl interactions in class II fingers. One bonded and two nonbonded models were studied. Two variant structures of the glucocorticoid receptor DNA-binding domain and a NMR structure from a fragment of methionyl-tRNA synthetase were subjected to long-time MD simulations with these models. Our analysis is focused on comparison with experimental and quantum mechanical data, concerning the local Zn-finger and overall structural and dynamic properties for these models. All models performed well, but the nonbonded models appeared to reproduce the protein dynamics in better agreement with experimental data than does the bonded description. 2001 John Wiley & Sons, Inc. Int J Quant Chem 83: 230,244, 2001 [source]

    The NMR structure of [Xd(C2)]4 investigated by molecular dynamics simulations

    Thrse E. Malliavin
    Abstract The i-motif tetrameric structure is built up from two parallel duplexes intercalated in a head-to-tail orientation, and held together by hemiprotonated cytosine pairs. Two topologies exist for the i-motif structure, one with outermost 3, extremities and the other with outermost 5, extremities, called the 3,E and 5,E topology, respectively. Since the comparison of sugar and phosphate group interactions between the two topologies is independent of the length of the intercalation motif, the relative stability of the 3,E and 5,E topologies therefore should not depend on this length. Nevertheless, it has been shown that the 3,E topology of the [d(C2)]4 is much more stable than the 5,E topology, and that the former is the only species observed in solution. In order to understand the reason for this atypical behavior, the NMR structure of the [Xd(C2)]4 was determined and analyzed by molecular dynamics simulations. In the NMR structure, the width of the narrow groove is slightly smaller than in previously determined i-motif structures, which supports the importance of phosphodiester backbone interactions in the structure stability. The simulations show that the stacking of cytosines, essential for the i-motif stability, is produced by a similar and non-negative twisting of the phosphodiester backbones. The twisting is induced by an interaction between the backbones; the [Xd(C2)]4 in 5,E topology, exhibiting very limited interaction between the phosphodiester backbones, is thus unstable. Copyright 2002 John Wiley & Sons, Ltd. [source]

    Current trends in the structure,activity relationship studies of the endogenous agouti-related protein (AGRP) melanocortin receptor antagonist

    Andrzej M. Wilczynski
    Abstract Agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin-3 and -4 (MC3R and MC4) G-protein coupled receptors. The 87,132 amino acid C-terminal domain of hAGRP possesses five disulfide bridges and a well-defined three-dimensional structure that displays full biological activity as compared to the full-length protein. Based on the NMR structure of the C-terminal AGRP(87,132), a novel mini-protein, referred to as "Mini-AGRP" was designed that exhibited receptor binding affinity and antagonism similar to that of the parent hAGRP(87,132) protein. It was demonstrated that this new-engineered protein autonomously folds to the inhibitor cystine knot (ICK) motif. As this AGRP is a novel mammalian protein involved in energy homeostasis and possibly other physiological functions remaining to be identified, structure-function studies are starting to emerge toward the understanding of how this unique protein putatively interacts with the melanocortin receptors with the objective of designing potential therapeutic agents for in vivo physiological studies. This article summarizes the progress to date of AGRP-based structure,activity relationships and putative ligand,receptor interactions. 2005 Wiley Periodicals, Inc. [source]

    NMR solution structure of KP-TerB, a tellurite-resistance protein from Klebsiella pneumoniae

    PROTEIN SCIENCE, Issue 4 2008
    Sheng-Kuo Chiang
    Abstract Klebsiella pneumoniae (KP), a Gram-negative bacterium, is a common cause of hospital-acquired bacterial infections worldwide. Tellurium (Te) compounds, although relatively rare in the environment, have a long history as antimicrobial and therapeutic agents. In bacteria, tellurite (TeO3,2) resistance is conferred by the ter (Ter) operon (terZABCDEF). Here, on the basis of 2593 restraints derived from NMR analysis, we report the NMR structure of TerB protein (151 amino acids) of KP (KP-TerB), which is mainly composed of seven ,-helices and a 310 helix, with helices II to V apparently forming a four-helix bundle. The ensemble of 20 NMR structures was well-defined, with a RMSD of 0.32 0.06 for backbone atoms and 1.11 0.07 for heavy atoms, respectively. A unique property of the KP-TerB structure is that the positively and negatively charged clusters are formed by the N-terminal positively and C-terminal negatively charged residues, respectively. To the best of our knowledge, the protein sequence and structures of KP-TerB are unique. [source]

    Solution structure of HI1506, a novel two-domain protein from Haemophilus influenzae

    PROTEIN SCIENCE, Issue 5 2007
    Nese Sari
    Abstract HI1506 is a 128-residue hypothetical protein of unknown function from Haemophilus influenzae. It was originally annotated as a shorter 85-residue protein, but a more detailed sequence analysis conducted in our laboratory revealed that the full-length protein has an additional 43 residues on the C terminus, corresponding with a region initially ascribed to HI1507. As part of a larger effort to understand the functions of hypothetical proteins from Gram-negative bacteria, and H. influenzae in particular, we report here the three-dimensional solution NMR structure for the corrected full-length HI1506 protein. The structure consists of two well-defined domains, an ,/, 50-residue N-domain and a 3-, 32-residue C-domain, separated by an unstructured 30-residue linker. Both domains have positively charged surface patches and weak structural homology with folds that are associated with RNA binding, suggesting a possible functional role in binding distal nucleic acid sites. [source]

    NMR structure of the pseudo -receiver domain of CikA

    PROTEIN SCIENCE, Issue 3 2007
    Tiyu Gao
    Abstract The circadian input kinase (CikA) is a major element of the pathway that provides environmental information to the circadian clock of the cyanobacterium Synechococcus elongatus. CikA is a polypeptide of 754 residues and has three recognizable domains: GAF, histidine protein kinase, and receiver-like. This latter domain of CikA lacks the conserved phospho-accepting aspartyl residue of bona fide receiver domains and is thus a pseudo -receiver (PsR). Recently, it was shown that the PsR domain (1) attenuates the autokinase activity of CikA, (2) is necessary to localize CikA to the cell pole, and (3) is necessary for the destabilization of CikA in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The solution structure of the PsR domain of CikA, CikAPsR, is presented here. A model of the interaction between the PsR domain and HPK portion of CikA provides a potential explanation for how the PsR domain attenuates the autokinase activity of CikA. Finally, a likely quinone-binding surface on CikAPsR is shown here. [source]

    NMR structure of the enzyme GatB of the galactitol-specific phosphoenolpyruvate-dependent phosphotransferase system and its interaction with GatA

    PROTEIN SCIENCE, Issue 10 2006
    Laurent Volpon
    Abstract The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate-specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol-specific transporter (IIgat) belongs to the glucitol family and is structurally the least well-characterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of the IIB subunit (GatB). GatB consists of a central four-stranded parallel ,-sheet flanked by ,-helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between ,1 and ,1 that folds into a P-loop-like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA-interacting residues. [source]

    High resolution structure of the HDGF PWWP domain: A potential DNA binding domain

    PROTEIN SCIENCE, Issue 2 2006
    Stephen M. Lukasik
    Abstract Hepatoma Derived Growth Factor (HDGF) is an endogenous nuclear-targeted mitogen that is linked with human disease. HDGF is a member of the weakly conserved PWWP domain family. This 70,amino acid motif, originally identified from the WHSC1 gene, has been found in more than 60 eukaryotic proteins. In addition to the PWWP domain, many proteins in this class contain known chromatin remodeling domains, suggesting a role for HDGF in chromatin remodeling. We have determined the NMR structure of the HDGF PWWP domain to high resolution using a combination of NOEs, J-couplings, and dipolar couplings. Comparison of this structure to a previously determined structure of the HDGF PWWP domain shows a significant difference in the C-terminal region. Comparison to structures of other PWWP domains shows a high degree of similarity to the PWWP domain structures from Dnmt3b and mHRP. The results of selected and amplified binding assay and NMR titrations with DNA suggest that the HDGF PWWP domain may function as a nonspecific DNA-binding domain. Based on the NMR titrations, we propose a model of the interaction of the PWWP domain with DNA. [source]

    Heterogeneity and dynamics in villin headpiece crystal structures

    Jianmin Meng
    The villin headpiece domain (HP67) is the C-terminal F-actin-binding motif that confers F-actin-bundling activity to villin, a component of the actin bundles that support the brush-border microvilli. It has been investigated extensively by both experimental and theoretical measurements. Our laboratory, for example, has determined both its NMR and its crystal structures. This study presents the structures of HP67 and its pH-stabilized mutant (H41Y) in a different crystal form and space group. For both constructs, two molecules are found in each asymmetric unit in the new space group P61. While one of the two structures (Mol A) is structurally similar to our previously determined structure (Mol X), the other (Mol B) has significant deviations, especially in the N-terminal subdomain, where lattice contacts do not appear to contribute to the difference. In addition, the structurally most different crystal structure, Mol B, is actually closer to the averaged NMR structure. Harmonic motions, as suggested by the B -factor profiles, differ between these crystal structures; crystal structures from the same space group share a similar pattern. Thus, heterogeneity and dynamics are observed in different crystal structures of the same protein even for a protein as small as villin headpiece. [source]

    The X-ray structure of a recombinant major urinary protein at 1.75, resolution.

    A comparative study of X-ray, NMR-derived structures
    Major urinary proteins belong to the lipocalin family and are present in the urine of rodents as an ensemble of isoforms with pheromonal activity. The crystal structure of a recombinant mouse MUP (rMUP) was solved by the molecular-replacement technique and refined to an R factor and Rfree of 20 and 26.5%, respectively, at 1.75, resolution. The structure was compared with an NMR model and with a crystallographic structure of the wild-type form of the protein. The crystal structures determined in different space groups present significantly smaller conformational differences amongst themselves than in comparison with NMR models. Some, but not all, of the conformational differences between the crystal and solution structures can be explained by the influence of crystallographic contacts. Most of the differences between the NMR and X-ray structures were found in the N-terminus and loop regions. A number of side chains lining the hydrophobic pocket of the molecule are more tightly packed in the NMR structure than in the crystallographic model. Surprisingly, clear and continuous electron density for a ligand was observed inside the hydrophobic pocket of this recombinant protein. Conformation of the ligand modelled inside the density is coherent with the results of recent NMR experiments. [source]

    A systematic case study on using NMR models for molecular replacement: p53 tetramerization domain revisited

    Yu Wai Chen
    Molecular replacement using search models derived from nuclear magnetic resonance (NMR) spectroscopy has often proved problematic. It has been known for some time that the overall differences in atomic positions (r.m.s.d.) between the crystalline and the solution states of the same protein are of the order of 1,2, and approach the limit of molecular replacement. In most cases, this structural difference is a result of calculating the NMR structure with insufficient data, yielding an NMR structure of limited accuracy. A systematic case study was performed to investigate the use of NMR models for molecular replacement on the p53 tetramerization domain: NMR search models of varying degrees of accuracy were employed to solve phases for the 1.5, X-ray diffraction data. An approximate correlation was found between the accuracy of the NMR search model and the clarity and quality of the molecular-replacement solution. It was found that ensemble models perform better than single averaged models and have a larger tolerance in model inaccuracy. Also, distance-derived B factors can improve the performance of single models. [source]

    Cover Picture: NMR Structure of the Single QALGGH Zinc Finger Domain from the Arabidopsis thaliana SUPERMAN Protein (ChemBioChem 2-3/2003)

    CHEMBIOCHEM, Issue 2-3 2003
    Carla Isernia Prof.
    Abstract The cover picture shows the NMR structure of the SUPERMAN zinc finger domain, which is the first high-resolution structure of a classical zinc finger domain from a plant protein. The structure consists of a very well-defined ,,, motif, typical of all the other Cys2,His2 zinc fingers so far structurally characterized. As a consequence, the QALGGH sequence, which is highly conserved in plant protein classical zinc finger domains, is located at the N terminus of the , helix. Interestingly, this domain region, in animal protein zinc fingers, is constituted of hypervariable residues deputed to the recognition of the DNA bases. Therefore, a peculiar DNA recognition code for the QALGGH zinc finger domain is proposed in the article by Fattorusso and co-workers on p. 171 ff. [source]

    Solution Structure of a DNA Duplex Containing a Biphenyl Pair

    Zeena Johar
    Abstract Hydrogen-bonding and stacking interactions between nucleobases are considered to be the major noncovalent interactions that stabilize the DNA and RNA double helices. In recent work we found that one or multiple biphenyl pairs, devoid of any potential for hydrogen bond formation, can be introduced into a DNA double helix without loss of duplex stability. We hypothesized that interstrand stacking interactions of the biphenyl residues maintain duplex stability. Here we present an NMR structure of the decamer duplex d(GTGACXGCAG), d(CTGCYGTCAC) that contains one such X/Y biaryl pair. X represents a 3,,,5,,-dinitrobiphenyl- and Y a 3,,,4,,-dimethoxybiphenyl C -nucleoside unit. The experimentally determined solution structure shows a B-DNA duplex with a slight kink at the site of modification. The biphenyl groups are intercalated side by side as a pair between the natural base pairs and are stacked head to tail in van der Waals contact with each other. The first phenyl rings of the biphenyl units each show tight intrastrand stacking to their natural base neighbors on the 3,-side, thus strongly favoring one of two possible interstrand intercalation structures. In order to accommodate the biphenyl units in the duplex the helical pitch is widened while the helical twist at the site of modification is reduced. Interestingly, the biphenyl rings are not static in the duplex but are in dynamic motion even at 294,K. [source]

    Synthesis and structural characterization of a mimetic membrane-anchored prion protein

    FEBS JOURNAL, Issue 6 2006
    Matthew R. Hicks
    During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP,GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP,GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP. [source]

    Three-dimensional structure of the histidine-containing phosphocarrier protein (HPr) from Enterococcus faecalis in solution

    FEBS JOURNAL, Issue 3 2001
    Till Maurer
    The histidine-containing phosphocarrier protein (HPr) transfers a phosphate group between components of the prokaryotic phosphoenolpyruvate-dependent phosphotransferase system (PTS), which is finally used to phosphorylate the carbohydrate transported by the PTS through the cell membrane. Recently it has also been found to act as an intermediate in the signaling cascade that regulates transcription of genes related to the carbohydrate-response system. Both functions involve phosphorylation/dephosphorylation reactions, but at different sites. Using multidimensional 1H-NMR spectroscopy and angular space simulated annealing calculations, we determined the structure of HPr from Enterococcus faecalis in aqueous solution using 1469 distance and 44 angle constraints derived from homonuclear NMR data. It has a similar overall fold to that found in HPrs from other organisms. Four , strands, A, B, C, D, encompassing residues 2,7, 32,37, 40,42 and 60,66, form an antiparallel , sheet lying opposite the two antiparallel , helices, a and c (residues 16,26 and 70,83). A short , helix, b, from residues 47,53 is also observed. The pairwise root mean square displacement for the backbone heavy atoms of the mean of the 16 NMR structures to the crystal structure is 0.164 nm. In contrast with the crystalline state, in which a torsion angle strain in the active-center loop has been described [Jia, Z., Vandonselaar, M., Quail, J.W. & Delbaere, L.T.J. (1993) Nature (London) 361, 94,97], in the solution structure, the active-site His15 rests on top of helix a, and the phosphorylation site N,1 of the histidine ring is oriented towards the surface, making it easily accessible to the solvent. Back calculation of the 2D NOESY NMR spectra from both the NMR and X-ray structures shows that the active-center structure derived by X-ray crystallography is not compatible with experimental data recorded in solution. The observed torsional strain must either be a crystallization artefact or represents a conformational state that exists only to a small extent in solution. [source]

    Isolation and X-Ray Structures of Reactive Intermediates of Organocatalysis with Diphenylprolinol Ethers and with Imidazolidinones

    5-Repulsion, A Survey, Comparison with Computed Structures, the Geminal-Diaryl Effect at Work, with 1-Acyl-imidazolidinones: The
    Abstract Reaction of 2-phenylacetaldehyde with the Me3Si ether of diphenyl-prolinol, with removal of H2O, gives a crystalline enamine (1). The HBF4 salts of the MePh2Si ether of diphenyl-prolinol and of 2-(tert -butyl)-3-methyl- and 5-benzyl-2,2,3-trimethyl-1,3-imidazolidin-4-one react with cinnamaldehyde to give crystalline iminium salts 2, 3, and 4. Single crystals of the enamine and of two iminium salts, 2 and 3, were subjected to X-ray structure analysis (Figs.,1, 2, and 6), and a 2D-NMR spectrum of the third iminium salt was recorded (Fig.,7). The crystal and NMR structures confirm the commonly accepted, general structures of the two types of reactive intermediates in organocatalysis with the five-membered heterocycles, i.e., D, E (Scheme,2). Fine details of the crystal structures are discussed in view of the observed stereoselectivities of the corresponding reactions with electrophiles and nucleophiles. The structures 1 and 2 are compared with those of other diphenyl-prolinol derivatives (from the Cambridge File CSD; Table,1) and discussed in connection with other reagents and ligands, containing geminal diaryl groups and being used in enantioselective synthesis (Fig.,4). The iminium ions 3 and 4 are compared with N -acylated imidazolidinones F and G (Figs.,9, 12, and 13, and Table,3), and common structural aspects such as minimalization of 1,5-repulsion (the ,A1,3 -effect'), are discussed. The crystal structures of the simple diphenyl-prolinol,HBF4 salt (Fig.,3) and of Boc- and benzoyl-(tert -butyl)methyl-imidazolidinone (Boc-BMI and Bz-BMI, resp.; Figs.,10 and 11) are also reported. Finally, the crystal structures are compared with previously published theoretical structures, which were obtained from high-level-of-theory DFT calculations (Figs.,5 and 8, and Table,2). Delicate details including pyramidalization of trigonal N-atoms, distortions around iminium CN bonds, shielding of diastereotopic faces, and the , -interaction between a benzene ring and a Me group match so well with, and were actually predicting the experimental results that the question may seem appropriate, whether one will soon start considering to carry out such calculations before going to the laboratory for experimental optimizations. [source]

    Cold-adapted signal proteins: NMR structures of pheromones from the antarctic ciliate Euplotes nobilii

    IUBMB LIFE, Issue 8-9 2007
    William J. Placzek
    Abstract Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear 1H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-,-helix bundle with an up-down-up topology and a 310-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters. [source]

    Closed loop folding units from structural alignments: Experimental foldons revisited

    Sree V. Chintapalli
    Abstract Nonoverlapping closed loops of around 25,35 amino acids formed via nonlocal interactions at the loop ends have been proposed as an important unit of protein structure. This hypothesis is significant as such short loops can fold quickly and so would not be bound by the Leventhal paradox, giving insight into the possible nature of the funnel in protein folding. Previously, these closed loops have been identified either by sequence analysis (conservation and autocorrelation) or studies of the geometry of individual proteins. Given the potential significance of the closed loop hypothesis, we have explored a new strategy for determining closed loops from the insertions identified by the structural alignment of proteins sharing the same overall fold. We determined the locations of the closed loops in 37 pairs of proteins and obtained excellent agreement with previously published closed loops. The relevance of NMR structures to closed loop determination is briefly discussed. For cytochrome c, cytochrome b562 and triosephophate isomerase, independent folding units have been determined on the basis of hydrogen exchange experiments and misincorporation proton-alkyl exchange experiments. The correspondence between these experimentally derived foldons and the theoretically derived closed loops indicates that the closed loop hypothesis may provide a useful framework for analyzing such experimental data. 2010 Wiley Periodicals, Inc. J Comput Chem, 2010 [source]

    Application of torsion angle molecular dynamics for efficient sampling of protein conformations

    Jianhan Chen
    Abstract We investigate the application of torsion angle molecular dynamics (TAMD) to augment conformational sampling of peptides and proteins. Interesting conformational changes in proteins mainly involve torsional degrees of freedom. Carrying out molecular dynamics in torsion space does not only explicitly sample the most relevant degrees of freedom, but also allows larger integration time steps with elimination of the bond and angle degrees of freedom. However, the covalent geometry needs to be fixed during internal coordinate dynamics, which can introduce severe distortions to the underlying potential surface in the extensively parameterized modern Cartesian-based protein force fields. A "projection" approach (Katritch et al. J Comput Chem 2003, 24, 254,265) is extended to construct an accurate internal coordinate force field (ICFF) from a source Cartesian force field. Torsion crossterm corrections constructed from local molecular fragments, together with softened van der Waals and electrostatic interactions, are used to recover the potential surface and incorporate implicit bond and angle flexibility. MD simulations of dipeptide models demonstrate that full flexibility in both the backbone ,/, and side chain ,1 angles are virtually restored. The efficacy of TAMD in enhancing conformational sampling is then further examined by folding simulations of small peptides and refinement experiments of protein NMR structures. The results show that an increase of several fold in conformational sampling efficiency can be reliably achieved. The current study also reveals some complicated intrinsic properties of internal coordinate dynamics, beyond energy conservation, that can limit the maximum size of the integration time step and thus the achievable gain in sampling efficiency. 2005 Wiley Periodicals, Inc. J Comput Chem 26: 1565,1578, 2005 [source]

    Structural studies and model membrane interactions of two peptides derived from bovine lactoferricin

    Leonard T. Nguyen
    Abstract The powerful antimicrobial properties of bovine lactoferricin (LfcinB) make it attractive for the development of new antimicrobial agents. An 11-residue linear peptide portion of LfcinB has been reported to have similar antimicrobial activity to lactoferricin itself, but with lower hemolytic activity. The membrane-binding and membrane-perturbing properties of this peptide were studied together with an amidated synthetic version with an added disulfide bond, which was designed to confer increased stability and possibly activity. The antimicrobial and cytotoxic properties of the peptides were measured against Staphylococcus aureus and Escherichia coli and by hemolysis assays. The peptides were also tested in an anti-cancer assay against neuroblastoma cell lines. Vesicle disruption caused by these LfcinB derivatives was studied using the fluorescent reporter molecule calcein. The extent of burial of the two Trp residues in membrane mimetic environments were quantitated by fluorescence. Finally, the solution NMR structures of the peptides bound to SDS micelles were determined to provide insight into their membrane bound state. The cyclic peptide was found to have greater antimicrobial potency than its linear counterpart. Consistent with this property, the two Trp residues of the modified peptide were suggested to be embedded deeper into the membrane. Although both peptides adopt an amphipathic structure without any regular ,-helical or -sheet conformation, the 3D-structures revealed a clearer partitioning of the cationic and hydrophobic faces for the cyclic peptide. Copyright 2004 European Peptide Society and John Wiley & Sons, Ltd. [source]

    Investigation of penetratin peptides Part 1.

    The environment dependent conformational properties of penetratin, two of its derivatives
    Abstract The homeodomain, the DNA-binding domain of Antennapedia homeoprotein, is composed of three ,-helices and one ,-turn between helices II and III. Its third helix from the N -terminal (helix III) can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. To the best of our knowledge, this helix III, called penetratin, which consists of 16 amino acids, is internalized by cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, the structure of penetratin was examined in both extracellular matrix-mimetic and membrane-mimetic environments; 1H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. The molecular conformations of two analogue peptides [(6,14-Phe)-penetratin and a 12 amino acid penetratin derivative (peptide 3)] were also studied. An atomic level comprehensive analysis of penetratin and its two analogues was performed. In a membrane-mimetic solvent system (TFEd2/water = 9 : 1), on the basis of 553 distance restraints, the 4,12 region of penetratin exhibits a bent, irregular helical structure on NMR examination. Interactions between hydrophobic amino acid residues in conjunction with H-bonds stabilize the secondary structure of the molecule. Thus, both derivatives adopt a helix-like conformation. However, while (6,14-Phe)-penetratin displays both ,-helical and 310 -helical features, the structure of peptide 3 is predominantly a 310 -helix. Of the three peptides, surprisingly (6,14-Phe)-penetratin has the largest helical content. An increase in the polarity of the molecular environment gradually disintegrates these helix-like secondary structures. In a highly aqueous molecular system (TFEd2/water = 1 : 9), the fast exchange of multiple conformers leads to too few distance restraints being extracted, therefore the NMR structures can no longer be determined. The NMR data show that only short-range order can be traced in these peptides. Under these conditions, the molecules adopt nascent helix-like structures. On the other hand, CD spectra could be recorded at any TFE/water ratio and the conformational interconversion could therefore be monitored as a function of the polarity of the molecular environment. The CD data were analysed comprehensively by the quantitative deconvolution method (CCA+). All three penetratin peptides display helical conformational features in a low dielectric medium, with significant differences as a function of their amino acid composition. However, these conformational features are gradually lost during the shift from an apolar to a polar molecular environment. Copyright 2002 European Peptide Society and John Wiley & Sons, Ltd. [source]

    From fundamental studies of sporulation to applied spore research

    Imrich Bark
    Summary Sporulation in the Gram-positive bacterium, Bacillus subtilis, has been used as an excellent model system to study cell differentiation for almost half a century. This research has given us a detailed picture of the genetic, physiological and biochemical mechanisms that allow bacteria to survive harsh environmental conditions by forming highly robust spores. Although many basic aspects of this process are now understood in great detail, including the crystal and NMR structures of some of the key proteins and their complexes, bacterial sporulation still continues to be a highly attractive model for studying various cell processes at a molecular level. There are several reasons for such scientific interest. First, some of the complex steps in sporulation are not fully understood and/or are only described by ,controversial' models. Second, intensive research on unicellular development of a single microorganism, B. subtilis, left us largely unaware of the multitude of diverse sporulation mechanisms in many other Gram-positive endospore and exospore formers. This diversity would likely be increased if we were to include sporulation processes in the Gram-negative spore formers. Spore formers have great potential in applied research. They have been used for many years as biodosimeters and as natural insecticides, exploited in the industrial production of enzymes, antibiotics, used as probiotics and, more, exploited as possible vectors for drug delivery, vaccine antigens and other immunomodulating molecules. This report describes these and other aspects of current fundamental and applied spore research that were presented at European Spores Conference held in Smolenice Castle, Slovakia, June 2004. [source]

    NMR solution structure of KP-TerB, a tellurite-resistance protein from Klebsiella pneumoniae

    PROTEIN SCIENCE, Issue 4 2008
    Sheng-Kuo Chiang
    Abstract Klebsiella pneumoniae (KP), a Gram-negative bacterium, is a common cause of hospital-acquired bacterial infections worldwide. Tellurium (Te) compounds, although relatively rare in the environment, have a long history as antimicrobial and therapeutic agents. In bacteria, tellurite (TeO3,2) resistance is conferred by the ter (Ter) operon (terZABCDEF). Here, on the basis of 2593 restraints derived from NMR analysis, we report the NMR structure of TerB protein (151 amino acids) of KP (KP-TerB), which is mainly composed of seven ,-helices and a 310 helix, with helices II to V apparently forming a four-helix bundle. The ensemble of 20 NMR structures was well-defined, with a RMSD of 0.32 0.06 for backbone atoms and 1.11 0.07 for heavy atoms, respectively. A unique property of the KP-TerB structure is that the positively and negatively charged clusters are formed by the N-terminal positively and C-terminal negatively charged residues, respectively. To the best of our knowledge, the protein sequence and structures of KP-TerB are unique. [source]

    Structure of the 21,30 fragment of amyloid ,-protein

    PROTEIN SCIENCE, Issue 6 2006
    Andrij Baumketner
    Abstract Folding and self-assembly of the 42-residue amyloid ,-protein (A,) are linked to Alzheimer's disease (AD). The 21,30 region of A,, A,(21,30), is resistant to proteolysis and is believed to nucleate the folding of full-length A,. The conformational space accessible to the A,(21,30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24,K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures. [source]

    Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements

    Charlotte A. Scarff
    The three-dimensional conformation of a protein is central to its biological function. The characterisation of aspects of three-dimensional protein structure by mass spectrometry is an area of much interest as the gas-phase conformation, in many instances, can be related to that of the solution phase. Travelling wave ion mobility mass spectrometry (TWIMS) was used to investigate the biological significance of gas-phase protein structure. Protein standards were analysed by TWIMS under denaturing and near-physiological solvent conditions and cross-sections estimated for the charge states observed. Estimates of collision cross-sections were obtained with reference to known standards with published cross-sections. Estimated cross-sections were compared with values from published X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy structures. The cross-section measured by ion mobility mass spectrometry varies with charge state, allowing the unfolding transition of proteins in the gas phase to be studied. Cross-sections estimated experimentally for proteins studied, for charge states most indicative of native structure, are in good agreement with measurements calculated from published X-ray and NMR structures. The relative stability of gas-phase structures has been investigated, for the proteins studied, based on their change in cross-section with increase in charge. These results illustrate that the TWIMS approach can provide data on three-dimensional protein structures of biological relevance. Copyright 2008 John Wiley & Sons, Ltd. [source]

    Structure of human desArg-C5a

    William J. Cook
    The anaphylatoxin C5a is derived from the complement component C5 during activation of the complement cascade. It is an important component in the pathogenesis of a number of inflammatory diseases. NMR structures of human and porcine C5a have been reported; these revealed a four-helix bundle stabilized by three disulfide bonds. The crystal structure of human desArg-C5a has now been determined in two crystal forms. Surprisingly, the protein crystallizes as a dimer and each monomer in the dimer has a three-helix core instead of the four-helix bundle noted in the NMR structure determinations. Furthermore, the N-terminal helices of the two monomers occupy different positions relative to the three-helix core and are completely different from the NMR structures. The physiological significance of these structural differences is unknown. [source]