Distance Geometry Research Articles

Conformational studies of [Abu3, 11]‐SFTI‐1, an analogue of the trypsin inhibitor isolated from sunflower seeds

With only 14 amino acid residues, the trypsin inhibitor SFTI-1 is the smallest naturally occurring serine proteinase inhibitor. It consists of two cyclic fragments (with head-to-tail cyclization and a disulfide bridge). In our previous paper, we showed that the removal of the disulfide bridge produced 2.4-fold lower activity. Here, we present the total conformational analysis of the [Abu(3, 11)]-SFTI-1 analog by means of 2D NMR spectroscopy in conjunction with theoretical methods. The peptide was synthesized by Fmoc SPPS. It was cyclized with PyBop and DIPEA in DMF. The NMR studies were performed in DMSO-d(6) at 303 K. Conformations of the peptide studied were calculated by the following three approaches: distance geometry (DG), molecular dynamics (MD) and determination of the statistical weights of conformations. The first two algorithms use a CHARMM force field, whereas the last uses an ECEPP/3 force field. Our calculations resulted in three sets of conformers with 7, 9 and 6 representatives, respectively. All our results were compared with published ones. It was found that the peptide has an ill-defined structure. Despite its conformational flexibility, the binding loop (3-11 fragment) displayed geometry similar to the corresponding fragments of the other SFTI-1 analogs and to the inhibitor itself. Furthermore, the peptide bond between the Ile7 and Pro8 residues adopts cis geometry, which is essential for inhibitory activity.

Optical Antenna Properties of Scanning Probe Tips: Plasmonic Light Scattering, Tip−Sample Coupling, and Near-Field Enhancement

The optical near-field distribution and enhancement near the apex of model scanning probe tips are calculated within the quasistatic approximation. The optical tip−sample coupling sensitively depends on both the tip and sample material. This, in addition to the tip−sample distance and apex geometry, is found to affect the spatial resolution that can be obtained in scattering near-field microscopy (s-SNOM). A pronounced structural plasmon resonant behavior is found for gold tips, which redshifts upon tip−sample approach on the length scale given by the tip radius. This near-field tip−sample coupling also allows for surface plasmon excitation in the sample. With the critical dimensions of the tip apex in the range of 10 to several 10s of nanometers, the results are found to be in good agreement with experiment and more rigorous theoretical treatments.

Electromagnetic impact welding of aluminum to stainless steel sheets

Welding of aluminum (Al) to stainless steel (SS) is difficult due to the difference in their melting temperatures. The processes, which are available for welding of Al-to-SS, are friction welding, laser welding, electric discharge welding, etc. The present study investigates electromagnetic impact welding of Al-to-SS by using Al as the driver sheet. The effects of process parameters such as energy, standoff distance and coil geometry on the tensile shear strength of the welds are examined. A study has been carried out to characterize the effectiveness of the process of welding 1 mm thick Al sheet to 0.25 mm thick SS sheet. The results of the microstructure and tensile shear strength tests are also reported.

Conformational Preference and Potential Templates ofN‐Methylated Cyclic Pentaalanine Peptides

Systematic N-methylation of all peptide bonds in the cyclic pentapeptide cyclo(-D-Ala-Ala(4)-) has been performed yielding 30 different N-methylated derivatives, of which only seven displayed a single conformation on the NMR time scale. The conformation of these differentially N-methylated peptides was recently reported by us (J. Am. Chem. Soc. 2006, 128, 15 164-15 172). Here we present the conformational characterization of nine additional N-methylated peptides from the previous library which are not homogeneous but exist as a mixture in which at least one conformation is preferred by over 80 %. The structures of these peptides are investigated employing various 2D-NMR techniques, distance geometry calculations and further refined by molecular dynamics simulations in explicit DMSO. The comparison of the conformation of these nine peptides and the seven conformationally homogeneous peptides allow us to draw conclusions regarding the influence of N-methylation on the peptide backbone of cyclic pentapeptide of the class cyclo(-D-Ala-Ala(4)-). Here we present the different conformational classes of the peptides arising from the definitive pattern of N-methylation which can eventually serve as templates for the design of bioactive peptides.

Structural Characterization of Neurokinin-3 Receptor Selective Peptide Agonist Scyliorhinin II Bound to DPC Micelles

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12–18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.

Total Structure Determination of Grassypeptolide, a New Marine Cyanobacterial Cytotoxin

A collection of the cyanobacterium Lyngbya confervoides off Grassy Key in Florida yielded grassypeptolide (1), a 31-membered macrocyclic depsipeptide with unusually high D-amino acid content, two thiazolines, and one beta-amino acid. We report the rigorous 3D structure determination and conformational analysis in solution and solid state by NMR, MS, X-ray crystallography, chemical degradation, and molecular modeling involving distance geometry and restrained molecular dynamics. Grassypeptolide (1) inhibited cancer cell growth with IC50 values from 1.0 to 4.2 microM.

A Branch‐and‐Prune algorithm for the Molecular Distance Geometry Problem

AbstractThe Molecular Distance Geometry Problem consists in finding the positions in of the atoms of a molecule, given some of the inter‐atomic distances. We show that under an additional requirement on the given distances this can be transformed to a combinatorial problem. We propose a Branch‐and‐Prune algorithm for the solution of this problem and report on very promising computational results.

Solving molecular distance geometry problems by global optimization algorithms

In this paper we consider global optimization algorithms based on multiple local searches for the Molecular Distance Geometry Problem (MDGP). Three distinct approaches (Multistart, Monotonic Basin Hopping, Population Basin Hopping) are presented and for each of them a computational analysis is performed. The results are also compared with those of two other approaches in the literature, the DGSOL approach (More, Wu in J. Glob. Optim. 15:219---234, 1999) and a SDP based approach (Biswas et al. in An SDP based approach for anchor-free 3D graph realization, Technical Report, Operations Research, Stanford University, 2005).

Structure and Electronic Spectra of DNA Mini-hairpins with Gn:CnStems

The solution structure of a synthetic DNA mini-hairpin possessing a stilbenediether linker and three G:C base pairs has been obtained using (1)H NMR spectral data and constrained torsion angle molecular dynamics. Notable features of this structure include a compact hairpin loop having a short stilbene-guanine plane-to-plane distance and approximate B-DNA geometry for the three base pairs. Comparison of the electronic spectra of mini-hairpins having one-to-four G:C base pairs and stilbenediether or hexamethyleneglycol linkers reveals the presence of features in the UV and CD spectra of the stilbene-linked hairpins that are not observed for the ethyleneglycol-linked hairpins. Investigation of the electronic structure of a stilbene-linked hairpin having a single G:C base pair by means of time-dependent density functional theory shows that the highest occupied molecular orbital, but not the lowest unoccupied molecular orbital, is delocalized over the stilbene and adjacent guanine. The calculated UV and CD spectra are highly dependent upon hairpin conformation, but reproduce the major features of the experimental spectra. These results illustrate the utility of an integrated experimental and theoretical approach to understanding the complex electronic spectra of pi-stacked chromophores.

Generating Conformer Ensembles Using a Multiobjective Genetic Algorithm

The task of generating a nonredundant set of low-energy conformations for small molecules is of fundamental importance for many molecular modeling and drug-design methodologies. Several approaches to conformer generation have been published. Exhaustive searches suffer from the exponential growth of the search space with increasing degrees of conformational freedom (number of rotatable bonds). Stochastic algorithms do not suffer as much from the exponential increase of search space and provide a good coverage of the energy minima. Here, the use of a multiobjective genetic algorithm in the generation of conformer ensembles is investigated. Distance geometry is used to generate an initial conformer, which is then subject to geometric modifications encoded by the individuals of the genetic algorithm. The geometric modifications apply to torsion angles about rotatable bonds, stereochemistry of double bonds and tetrahedral chiral centers, and ring conformations. The geometric diversity of the evolving conformer ensemble is preserved by a fitness-sharing mechanism based on the root-mean-square distance of the atomic coordinates. Molecular symmetry is taken into account in the distance calculation. The geometric modifications introduce strain into the structures. The strain is relaxed using an MMFF94-like force field in a postprocessing step that also removes conformational duplicates and structures whose strain energy remains above a predefined window from the minimum energy value found in the set. The implementation, called Balloon, is available free of charge on the Internet ( http://www.abo.fi/~mivainio/balloon/).

Further evidence for a C-terminal structural motif in CCK 2 receptor active peptide hormones

A comparison of the conformational characteristics of the related hormones [Nle 15] gastrin-17 and [Tyr 9-SO 3] cholecystokinin-15, in membrane-mimetic solutions of dodecylphosphocholine micelles and water, was undertaken using NMR spectroscopy to investigate the possibility of a structural motif responsible for the two hormones common ability to stimulate the CCK 2 receptor. Distance geometry calculations and NOE-restrained molecular dynamics simulations in biphasic solvent boxes of decane and water pointed to the two peptides adopting near identical helical C-terminal configurations, which extended one residue further than their shared pentapeptide sequence of Gly-Trp-Met-Asp-Phe-NH 2. The C-terminal conformation of [Nle 15] gastrin-17 contained a short α-helix spanning the Ala 11-Trp 14 sequence and an inverse γ-turn centered on Nle 15 while that of [Tyr 9-SO 3] cholecystokinin-15 contained a short 3 10 helix spanning its Met 10 to Met 13 sequence and an inverse γ-turn centered on Asp 14. Significantly, both the C-terminal helices were found to terminate in type I β-turns spanning the homologous Gly-Trp-Met-Asp sequences. This finding supports the hypothesis that this structural motif is a necessary condition for CCK 2 receptor activation given that both gastrin and cholecystokinin have been established to follow a membrane-associated pathway to receptor recognition and activation. Comparison of the conformations for the non-homologous C-terminal tyrosyl residues of [Nle 15] gastrin-17 and [Tyr 9-SO 3] cholecystokinin-15 found that they lie on opposite faces of the conserved C-terminal helices. The positioning of this tyrosyl residue is known to be essential for CCK 1 activity and non-essential for CCK 2 activity, pointing to it as a possible differentiator in CCK 1/CCK 2 receptor selection. The different tyrosyl orientations were retained in molecular models for the [Nle 15] gastrin-17/CCK 2 receptor and [Tyr 9-SO 3] cholecystokinin-15/CCK 1 receptor complexes, highlighting the role of this residue as a likely CCK 1/CCK 2 receptor differentiator.

Double variable neighbourhood search with smoothing for the molecular distance geometry problem

We discuss the geometrical interpretation of a well-known smoothing operator applied to the Molecular Distance Geometry Problem (MDGP), and we then describe a heuristic approach based on Variable Neighbourhood Search on the smoothed and original problem. This algorithm often manages to find solutions having higher accuracy than other methods. This is important as small differences in the objective function value may point to completely different 3D molecular structures.

Rigid versus unique determination of protein structures with geometric buildup

We introduce a geometric buildup approach to the distance geometry problem in protein modeling, and discuss the necessary and sufficient conditions on the distances for rigid or unique determination of a protein structure. We describe a new buildup algorithm for determining protein structures rigidly instead of uniquely. The algorithm requires even fewer distance constraints than the general buildup algorithm. We present the test results from applying the algorithm to determining the protein structures with varying degrees of availability of the distances, and show that the new development increases the modeling ability of the geometric buildup method even more while retaining much of the computational feasibility of the method.

Evidence for a C-terminal structural motif in gastrin and its bioactive fragments in membrane mimetic media

The conformational preferences of human little gastrin, [Nle 15] gastrin-17, and its short analogues, gastrin-4 and [β-Ala 1] gastrin-5, which include the C-terminal tetrapeptide sequence Trp–Met–Asp–Phe–NH 2 crucial for gastrin bioactivity, were determined by NMR spectroscopy in aqueous solutions of zwitterionic dodecylphosphocholine micelles. Backbone HN chemical shift temperature variance, Hα chemical shift deviations and complex non-sequential NOE patterns pointed to the C-terminal of [Nle 15] gastrin-17 adopting an ordered conformation. Distance geometry calculations and NOE-restrained molecular dynamics simulations in membrane mimetic solvent boxes of decane and water indicated the C-terminal tetrapeptide sequence of all three peptides adopted a similar, well defined structure, with a general type IV β-turn observed for all three peptides. The conformation of [Nle 15] gastrin-17 consisted of two short helices between Leu 5–Glu 9 and Ala 11–Trp 14, with the one helix terminating in a type I β-turn spanning Gly 13–Asp 16. The experimental evidence and conformational characteristics of the three peptides in micellar media support a membrane-associated mechanism of receptor recognition and activation for the gastrin hormone family and furthermore point to a possible biologically relevant structural motif for gastrin activity.

Development of a voxel phantom of Japanese adult male in upright posture

A Japanese voxel phantom in upright posture, JM2, has been developed on the basis of CT images of a healthy Japanese adult male. Body characteristics of JM2 were compared with those of the supine voxel phantom, JM, previously developed using CT images of the same person. Differences were found in the shapes of the spine and lower abdomen and the locations of several organs such as kidneys, liver and stomach between the two phantoms. Specific absorbed fractions (SAFs) for 24 target and 11 sources organs were calculated for monoenergetic photon ranging from 0.01 to 4 MeV. It was found that the SAFs for the kidneys as source organ and the lower large intestine wall as target organ in JM2 were significantly higher than those in JM for all photon energies. The differences of the SAFs between the two phantoms were attributed to the differences in the organ distance and organ geometry depending on the posture.

Unique helical conformation of the fourth cytoplasmic loop of the CB 1 cannabinoid receptor in a negatively charged environment

The proximal portion of the C-terminus of the CB 1 cannabinoid receptor is a primary determinant for G-protein activation. A 17 residue proximal C-terminal peptide (rodent CB1 401–417), the intracellular loop 4 (IL4) peptide, mimicked the receptor’s G-protein activation domain. Because of the importance of the cationic amino acids to G-protein activation, the three-dimensional structure of the IL4 peptide in a negatively charged sodium dodecyl sulfate (SDS) micellar environment has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy and distance geometry calculations. Unambiguous proton NMR assignments were carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints were used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures which were refined using restrained energy minimization and dynamics. In water, the IL4 peptide prefers an extended conformation, whereas in SDS micelles, 3 10-helical conformation is induced. The predominance of 3 10-helical domain structure in SDS represents a unique difference compared with structure in alternative environments, which can significantly impact global electrostatic surface potential on the cytoplasmic surface of the CB 1 receptor and might influence the signal to the G-proteins.

The solution structure of horseshoe crab antimicrobial peptide tachystatin B with an inhibitory cystine‐knot motif

Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 A, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin.

Secondary structure inducing potential of β‐amino acids: Torsion angle clustering facilitates comparison and analysis of the conformation during MD trajectories

While numerous examples of beta-peptides--exclusively composed of beta-amino acids--have been investigated during the past decade, there are only few reports on the conformational preference of a single beta-amino acid when incorporated into a cyclopeptide. The conformational bias of beta-amino acids on the secondary structure of cyclopeptides has been investigated by NMR spectroscopy in combination with distance geometry (DG) and molecular dynamics (MD) calculations using experimental constraints. The atomic coordinate RMSD criterion usually employed for clustering of conformations after DG and MD calculations does not necessarily group similar peptide conformations, as there is an insufficient correlation between atomic coordinates and torsion angles. To improve on this shortcoming and to eliminate any arbitrary decisions during this process, a torsion angle clustering procedure has been implemented. For the cyclic pentapeptides cyclo-(-Val-beta-Hala-Phe-Leu-Ile-) 1 and cyclo-(-Ser-Pro-Leu-beta-Hasn-Asp-) 3, the beta-amino acid is found in the central position of an extended gamma-turn (pseudo gamma-turn, Psigamma-turn), while the beta-Hpro residue in the cyclic hexapeptide cyclo-(-Ser-beta-Hpro-Leu-Asn-Ile-Asp-) 5 preferentially occupies position i+1 of a pseudo beta-turn (Psibeta-turn). These results further corroborate the hypothesis of beta-amino acids being reliable inducers of secondary structure in cyclic penta- and hexapeptides. They can be employed in the de novo design of biologically active cyclopeptides in pharmaceutical research, since the three-dimensional presentation of pharmacophoric groups in the side chains can be tailored by incorporation of beta-amino acids in strategic sequential positions.

Sordarin Derivatives Induce a Novel Conformation of the Yeast Ribosome Translocation Factor eEF2

The sordarins are fungal specific inhibitors of the translation factor eEF2, which catalyzes the translocation of tRNA and mRNA after peptide bond formation. We have determined the crystal structures of eEF2 in complex with two novel sordarin derivatives. In both structures, the three domains of eEF2 that form the ligand-binding pocket are oriented in a different manner relative to the rest of eEF2 compared with our previous structure of eEF2 in complex with the parent natural product sordarin. Yeast eEF2 is also shown to bind adenylic nucleotides, which can be displaced by sordarin, suggesting that ADP or ATP also bind to the three C-terminal domains of eEF2. Fusidic acid is a universal inhibitor of translation that targets EF-G or eEF2 and is widely used as an antibiotic against Gram-positive bacteria. Based on mutations conferring resistance to fusidic acid, cryo-EM reconstructions, and x-ray structures of eEF2, EF-G, and an EF-G homolog, we suggest that the conformation of EF-G stalled on the 70 S ribosome by fusidic acid is similar to that of eEF2 trapped on the 80 S ribosome by sordarin.

Structured to reduce the mitogenicity of anti-CD3 antibody based on computer-guided molecular design

The mouse anti-human CD3 monoclonal antibody such as OKT3 is a potent immunosuppressive agent used in clinical transplantation to manipulate T-cell functions and prevent acute allograft rejection. However, the broad use of anti-CD3 antibody in clinical treatment was severely limited by the side effects of human anti-mouse antibody response and cytokine release syndrome. In this study, on the basis of a murine anti-human CD3 antibody yCD3 obtained in our previous work, a novel engineered anti-human CD3 antibody fragment ( i.e. V H-Linker-V L-Hinge-CH 3) was constructed with computer-guided molecular design method to avoid the clinical side effects. According to the distance geometry and intra-molecular interaction, the hinge region was re-designed and different from the parental hinge region in human IgG1. With the novel hinge region, the cysteine residues in hinge were exposure and prone to form the disulfide bond. Therefore, a novel bivalent antibody fragment named as mini-yCD3 was obtained. Mini-yCD3 displayed similar antigen-binding affinity and specificity to yCD3. Importantly, mini-yCD3 was shown to be much less potent in the induction of T-cell proliferation, cytokine release (interferon-γ and interleukin-2) and early activation marker expression on the cell surface (CD69 and CD25) than parental yCD3. Furthermore, mini-yCD3 was effective in modulating T-cell receptor/CD3 and inhibiting mixed lymphocyte reaction with similarity as yCD3. In conclusion, the constructed mini-yCD3 was much less mitogenic to T cells but retained potent immunosuppression, suggesting it might be an alternative to yCD3 as an immunosuppressive drug with less immunogenicity and toxicity for clinical application.