Well-defined Turn Research Articles

Measurements of beam characteristics on a 70 MeV H− cyclotron system for ISOL beam production

A 70 MeV H− cyclotron system was commissioned in order to measure the beam characteristics for the isotope separation on line (ISOL) operation in the energies range between 40 and 70 MeV. Using internal beams, the cyclotron magnet was precisely isochronized by applying the Smith-Garren method, achieving a main-coil current margin of 0.2 A for the beam stability. In the center region, beam profiles were measured by a differential radial probe to verify the specified dee voltage of 50 kV, which allows well-defined turn separation. Extracted beams were then used to check the beam line alignment by monitoring beam losses on segmented collimators and measuring variations on beam profiles. We measured transverse emittances of the cyclotron beam at a current of 25 µA by observing the beam profiles with upstream quadrupole strengths varied, which was the first time for this kind of 70 MeV cyclotron. Transmission efficiency along the beam line was measured as above 98% at a current of 100 µA. A temporary beam line was installed in the ISOL area, and we measured beam current distributions using a beam profile monitor shaped by wobbling at 60 Hz at a target location for beam diameters of 2 and 5 cm. A specific current distribution is generally required to reduce the maximum thermal stresses induced on the target. Finally, a maximum beam power of 50 kW was successfully tested at 70 MeV for 6 h.

An Artificial β-Sheet That Dimerizes through Parallel β-Sheet Interactions

This Article introduces a simple chemical model of a beta-sheet (artificial beta-sheet) that dimerizes by parallel beta-sheet formation in chloroform solution. The artificial beta-sheet consists of two N-terminally linked peptide strands that are linked with succinic or fumaric acid and blocked along one edge with a hydrogen-bonding template composed of 5-aminoanisic acid hydrazide. The template is connected to one of the peptide strands by a turn unit composed of (S)-2-aminoadipic acid (Aaa). 1H NMR spectroscopic studies show that these artificial beta-sheets fold in CDCl3 solution to form well-defined beta-sheet structures that dimerize through parallel beta-sheet interactions. Most notably, all of these compounds show a rich network of NOEs associated with folding and dimerization. The compounds also exhibit chemical shifts and coupling constants consistent with the formation of folded dimeric beta-sheet structures. The aminoadipic acid unit shows patterns of NOEs and coupling constants consistent with a well-defined turn conformation. The present system represents a significant step toward modeling the type of parallel beta-sheet interactions that occur in protein aggregation.

Peptide analogs containing the pentacyclo[5,4,0,02,6,03,6,05,9]undecane scaffold: conformational analysis in solution

In order to evaluate the conformational preferences of depsipeptides attached to polycyclic compounds, we have investigated conformational preferences in solution by NMR spectroscopy and in the theoretical domain by dynamic simulation, semi-empirical and ab initio calculations. In CDCl 3 solution, the polycyclic scaffolds 8 and 10 displayed different types of turn conformation while compounds 9, 11, (±)- 12 and (±)- 13 displayed a well-defined turn structure, characterized, respectively, by a eight-membered hydrogen-bonded ring and by a γ-like strand hydrogen-bonded ring for each peptide chain. In a strong solvating medium such as DMSO-d6 the depsipeptide (±)- 12 adopts a γ-like strand hydrogen-bonded ring for one peptide chain and, for the other one, a bifurcated H-bond formed by a γ-like strand and a non-classical C5-structure. Theoretical calculations, supported by the experimental data, made it possible to identify these conformations and were of great importance in the discussion of the observed results.

NMR studies on turn mimetic analogs derived from melanocyte-stimulating hormones.

Oligomers with alpha-aminooxy acids are reported to form very stable turn and helix structures, and they are supposed to be useful peptidomimetics for drug design. A recent report suggested that homochiral oxa-peptides form a strong eight-member-ring structure by a hydrogen bond between adjacent aminooxy-acid residues in a CDCl3 solution. In order to design an alpha-MSH analog with a stable turn conformation, we synthesized four tetramers and one pentamer, based on alpha-MSH sequence, and determined the solution structures of the molecules by two-dimensional NMR spectroscopy and simulated annealing calculations. The solution conformations of the three peptidomimetic molecules (TLV, TDV, and TLL) in DMSO-d6 contain a stable 7-membered-ring structure that is similar to a gamma-turn in normal peptides. Newly-designed tetramer TDF and pentamer PDF have a ball-type rigid structure that is induced by strong hydrogen bonds between adjacent amide protons and carbonyl oxygens. In conclusion, the aminooxy acids, easily prepared from natural or unnatural amino acids, can be employed to prepare peptidomimetic analogues with well-defined turn structures for pharmaceutical interest.

Interaction of a peptide from the receptor-binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: changes in backbone dynamics induced by binding.

The C-terminal receptor-binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N NMR relaxation experiments of the (15)N-labeled recombinant PAK pilin peptide in complex with an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus, were performed in order to probe for changes in the mobilities and dynamics of the peptide backbone as a result of antibody binding. The major results of these studies are as follows: binding of Fab leads to the preferential ordering of the first turn over the second turn in each isomer, binding of Fab partially stabilizes peptide backbone regions undergoing slow (microsecond to millisecond) exchange-related motions, and binding of Fab leads to a greater loss in backbone conformational entropy at pH 7.2 versus pH 4.5. The biological implications of these results will be discussed in relation to the role that fast and slow backbone motions play in PAK pilin peptide immunogenicity and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.

An unnatural amino acid that induces beta-sheet folding and interaction in peptides.

This paper introduces a unique amino acid that can readily be incorporated into peptides to make them fold into beta-sheetlike structures that dimerize through beta-sheet interactions. This new amino acid, Orn(i-PrCO-Hao), consists of an ornithine residue with the beta-strand-mimicking amino acid Hao [J. Am. Chem. Soc. 2000, 122, 7654-7661] attached to its side chain. When Orn(i-PrCO-Hao) is incorporated into a peptide, or appended to its N-terminus, the Hao group hydrogen bonds to the three subsequent residues to form a beta-sheetlike structure. The amino acid Orn(i-PrCO-Hao) is readily used in peptide synthesis as its Fmoc derivative, Fmoc-Orn(i-PrCO-Hao)-OH (3). Fmoc-Orn(i-PrCO-Hao)-OH behaves like a regular amino acid in peptide synthesis and was uneventfully incorporated into the peptide o-anisoyl-Val-Orn(i-PrCO-Hao)-Phe-Ile-Leu-NHMe (4) through standard automated Fmoc solid-phase peptide synthesis, with DIC and HOAt as the coupling agent for Fmoc-Orn(i-PrCO-Hao)-OH and o-anisic acid and HATU as the coupling agent for all other couplings. A second synthetic strategy was developed to facilitate the preparation of peptides with N-terminal Orn(i-PrCO-Hao) residues, which avoids the need for the preparation of Fmoc-Orn(i-PrCO-Hao)-OH. In this strategy, Boc-Orn(Fmoc)-OH is used as the penultimate amino acid in the peptide synthesis, and i-PrCO-Hao-OH (2) is used as the final amino acid. N-Terminal Orn(i-PrCO-Hao) peptide H-Orn(i-PrCO-Hao)-Phe-Ile-Leu-NHMe.TFA (5) was prepared in a fashion similar to that for 4, using DIC and HOAt as the coupling agent for i-PrCO-Hao-OH and HATU as the coupling agent for all other couplings. 1H NMR transverse-ROESY, coupling constant, and chemical shift studies establish that peptide 4 forms a dimeric beta-sheetlike structure in CDCl3 solution. The 1H NMR studies also suggest that the ornithine unit adopts a well-defined turn conformation. Analogous 1H NMR studies of peptide 5 indicate that this TFA salt folds but does not dimerize in CD3OD solution. Collectively, these synthetic and spectroscopic studies establish that the amino acid Orn(i-PrCO-Hao) induces beta-sheet structure and interactions in peptides in suitable organic solvents. Unlike the Hao amino acid, which acts as a prosthetic to replace three residues of the peptide strand, the Orn(i-PrCO-Hao) amino acid acts as a splint that helps enforce a beta-sheetlike structure without replacing the residues and their side chains. This feature of Orn(i-PrCO-Hao) is important, because it allows the creation of beta-sheet structure with minimal perturbation of the peptide sequence.

Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: conformation of the bound peptide.

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus. The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions. These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology. More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P. aeruginosa.

Probing the topological arrangement of the N- and C-terminal residues of bradykinin for agonist activity at the B1 receptor.

The conformational features of H-Lys-Arg-Ado-Ser-Pro-Phe-OH (Ado = 12-aminododecanoic acid), a des-Arg(9) analogue of Lys-bradykinin, have been determined by high-resolution NMR in the presence of a zwitterionic lipid environment. The analogue is the most active member of a series of analogues designed to probe the topological arrangement of the N- and C-termini required for agonistic activity at the B1 kinin receptor. A novel computational procedure for the utilization of NOE constraints from cis and trans configurational isomers is illustrated. Only with this computational methodology could the structural features of the N-terminus of the peptide be determined. Using radical-induced relaxation of the (1)H NMR signals, we measured the topological orientation of the peptide with respect to the zwitterionic lipid interface. The results indicate that the long, alkyl chain of the Ado amino acid imbeds into the lipid surface. The structural features of the C-terminus of the B1-selective analogue consist of a well-defined turn. Although removed from a standard beta-turn, required for activity at the B2 kinin receptor, the topological orientation of the side chains of the des-Arg(9) compound are surprisingly similar to those previously observed for beta-turn-containing bradykinin analogues. Therefore, we attribute the high B1 receptor selectivity, observed upon removal of Arg(9) from bradykinin, solely to the loss of a charged amino acid and not to altered structural features.

Binding domain of human parathyroid hormone receptor: from conformation to function.

A 31 amino acid fragment of the extracellular N-terminus of the human G-protein coupled receptor for parathyroid hormone (PTH1R) has been structurally characterized by NMR and molecular dynamics simulations. The fragment PTH1R[168-198] includes residues 173-189, shown by photoaffinity cross-linking to be a contact domain with position 13 of parathyroid hormone (PTH). The structure of PTH1R[168-198], determined in a micellar solution of dodecylphosphocholine to mimic the membrane environment, consists of three alpha-helices, separated by a well-defined turn and a flexible region. The topological orientation of PTH1R[168-198] was determined from nitroxide-radical induced relaxation of NMR signals utilizing 5- and 16-doxylstearic acid. The C-terminal helix (residues 190-196), consisting of seven amino acids of the first transmembrane domain, is very hydrophobic and embedded in the lipid core. This helix is preceded by a well-defined turn, forming an approximate 90 degrees bend, placing the other helices (residues 169-176 and 180-189), both of which are amphipathic, on the surface of the micelle. In this orientation, many hydrophilic residues of the receptor, including Glu177, Arg179, Arg181, Glu182, Asp185, and Arg186, are projecting toward the solvent available to form complementary Coulombic interactions with the polar residues of the principal binding domain of the ligand (e.g., Arg25, Lys26, Lys27, Asp30, and His32). Given that the binding domain of PTH adopts an amphipathic alpha-helix which lies on the membrane, we visualize ligand binding as a two stage process involving a nonspecific hydrophobic interaction of amphipathic helices with the membrane, followed by two-dimensional diffusion leading to highly specific, ligand-receptor interaction.

Three-dimensional structure and position of porcine motilin in sodium dodecyl sulfate micelles determined by 1H NMR.

The solution structure of the porcine gastrointestinal peptide hormone motilin was determined in the presence of sodium dodecyl sulfate (SDS) micelles at 28 degrees C using 1H nuclear magnetic resonance, full relaxation matrix analysis, and structure calculations based on restrained molecular dynamics. The structure of motilin in SDS micelles is described by a reverse gamma-turn and a beta-turn of type II in the N terminal end, an alpha-helical region in the middle of the molecule, and an extended structure at the C terminus. The position of the motilin molecule relative to the SDS micelles was probed by adding spin-labeled stearic acids, containing 12-doxyl or 5-doxyl spin-labels. We observed selective broadening of the proton resonances of residues 3-5 and concluded that they must be located in the interior of the micelle. These experiments suggest a structural model in which the hydrophobic N terminus consists of two well-defined turns buried in the interior of the micelle, whereas the amphiphilic alpha-helical part is located at the surface of the micelle. Spectral density mapping using a 13C label on the alphaC of Leu10 gave overall rotational correlation times taum of 6.6 and 4.5 ns at 35 and 45 degrees C, respectively. The long correlation time in combination with a high order parameter (S = 0.92) indicates that motilin has a rigid structure in the complex with the SDS micelle.

Pseudosubstrate Flexibility in Chicken Smooth Muscle Myosin Light Chain Kinase

Chicken smooth muscle myosin light chain kinase pseudosubstrate is contained within the C-terminal region of the sequence MLCK(774-807)amide. It forms a helical conformation which interacts with the connecting peptide contained within the N-terminal portion of the sequence. These regions are linked by a well-defined turn around Ser-787 which supports a model of pseudosubstrate regulation involving the hinging of the pseudosubstrate or autoregulatory recognition sequence away from the active site of the catalytic core following the binding of calmodulin to the kinase.

Complete sequential proton and nitrogen-15 nuclear magnetic resonance assignments and solution secondary structure of the blue copper protein azurin from Pseudomonas aeruginosa

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14,000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH 5.5 and 40 degrees C by using homo- and heteronuclear two-dimensional (2D) and three-dimensional (3D) nuclear magnetic resonance spectroscopic experiments. Combined analysis of a 3D homonuclear 1H Hartmann-Hahn nuclear Overhauser (3D 1H HOHAHA-NOESY) spectrum and a 3D heteronuclear 1H nuclear Overhauser 1H[15N] single-quantum coherence (3D 1H[15N] NOESY-HSQC) spectrum proved especially useful. The latter spectrum was recorded without irradiation of the water signal and provided for differential main chain amide (NH) exchange rates. NMR data were used to determine the secondary structure of azurin in solution. Comparison with the secondary structure of azurin obtained from X-ray analysis shows a virtually complete resemblance; the two beta-sheets and a 3(10)-alpha-3(10) helix are preserved at 40 degrees C, and most loops contain well-defined turns. Special findings are the unexpectedly slow exchange of the Asn-47 and Phe-114 NH's and the observation of His-46 and His-117 N epsilon 2H resonances. The implications of these observations for the assignment of azurin resonance Raman spectra, the rigidity of the blue copper site, and the electron transfer mechanism of azurin are discussed.

Three-dimensional structure of a complement control protein module in solution

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a β-sandwich arrangement; one face made up of three β-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate β-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the β-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

A DNA-binding homeodomain in histone H1

The structure of the globular domain of chicken histone H1 was compared here with that of the DNA-binding homeodomain in the Drosophila Antp protein, and they were observed to display considerable similarity. Both of them consist of three or four α-helices separated by well-defined turns. Charged residues in the aminoterminal end of α3 are therefore suggested to be responsible for sequence-specific recognition of DNA by the histone. In addition, α2 of H1, with a short leucine zipper in it, may be capable of protein-protein interaction in a similar manner to the other homeodomains.

Conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance.

The three-dimensional structure of recombinant desulfatohirudin in aqueous solution was determined by 1H nuclear magnetic resonance at 600 MHz and distance geometry calculations with the program DISMAN. The input for the structure calculations was prepared on the basis of complete sequence-specific resonance assignments at pH 4.5 and 22 degrees C and consisted of 425 distance constraints from nuclear Overhauser enhancements and 159 supplementary constraints from spin-spin coupling constants and from the identification of intramolecular hydrogen bonds. Residues 3-30 and 37-48 form a molecular core with two antiparallel beta-sheets and several well-defined turns. The three disulfide bonds 6-14, 16-28, and 22-39 were identified by NMR. In contrast to this well-defined molecular core, with an average root mean square distance for the polypeptide backbone of 0.8 A for a group of nine DISMAN solutions, no preferred conformation was found for the C-terminal segment 49-65, and a loop consisting of residues 31-36 is not uniquely constrained by the NMR data either. These structural properties of recombinant desulfatohirudin coincide closely with the previously described solution conformation of natural hirudin, but the presence of localized differences is indicated by chemical shift differences for residues Asp 5, Ser 9, Leu 15, Asp 53, Gly 54, and Asp 55.