Computational study of the conformational preferences of the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide.

alpha-Amino acids are important building blocks for the synthesis of a large number of bioactive compounds and pharmaceutical drugs. However, a literature survey revealed that no theoretical conformational study of alpha-amino acids with cage carbon frameworks has been performed to date. This paper reports the results of a conformational study on the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide (cage monopeptide), using molecular mechanics and ab initio methods. The in vacuo Ramachandran maps computed using the different parameterizations of the AMBER force field show the C7eq structure as the most favourable conformation, in contrast to the C7ax structure, that is the lowest energy conformation at the ab initio level. Analysis of these maps reveals the helical preference for the monopeptide and provides the potential for the cage residue to be incorporated into constrained peptide analogues.     

Conformational investigation of alpha, beta-dehydropeptides. XI. Molecular and crystal structure of Ac-(Z)-deltaPhe-NMe2 as compared to those of related molecules.

A series of three homologous dimethyldiamides Ac-(Z)-deltaPhe-NMe2, Ac-L-Phe-NMe2 and Ac-DL-Phe-NMe2 have been synthesized and their structures determined from single-crystal X-ray diffraction data. To learn more about the conformational preferences of the compounds studied, the fully relaxed phi, psi conformational energy maps on the free molecules of Ac-deltaAla-NMe2 and Ac-(Z)-deltaPhe-NMe2 were obtained with the HF/3-21G method and the calculated minima re-optimized with the DFT/B3LYP/6-31G** method. The crystal state results have been compared with the literature data. The studied dimethyldiamide Ac-deltaXaa-NMe2 combines the double bond in positions alpha, beta and the C-terminal tertiary amide within one molecule. As the representative probe with deltaXaa = deltaAla, (Z)-deltaLeu and (Z)-deltaPhe shows, in the solid state they adopt the conservative conformation with phi, psi approximately -45 degrees, approximately 130 degrees and with a non-planar tertiary amide bond, whatever the packing forces are. This conformation is located on the Ramachandran map in region H/F, which is of high-energy for common amino acids, but not so readily accessible to them. The free molecule calculations on Ac-deltaAla-NMe2 and Ac-(Z)-deltaPhe-NMe2 reveal that, in spite of dissimilar overall conformational profiles of these molecules, this structure is one of their low-energy conformers and for Ac-(Z)-deltaPhe-NMe2 it constitutes the global minimum. So, the theoretical results corroborate those experimental results proving that this structure is robust enough to avoid conformational distortion due to packing forces. In contrast to Ac-deltaXaa-NMe2, the saturated Ac-L/DL-Xaa-NMe2 shows the constancy of the associative patterns but do not prefer any molecular structure in the solid state.     

Distance geometry generates native-like folds for small helical proteins using the consensus distances of predicted protein structures.

For successful ab initio protein structure prediction, a method is needed to identify native-like structures from a set containing both native and non-native protein-like conformations. In this regard, the use of distance geometry has shown promise when accurate inter-residue distances are available. We describe a method by which distance geometry restraints are culled from sets of 500 protein-like conformations for four small helical proteins generated by the method of Simons et al. (1997). A consensus-based approach was applied in which every inter-Calpha distance was measured, and the most frequently occurring distances were used as input restraints for distance geometry. For each protein, a structure with lower coordinate root-mean-square (RMS) error than the mean of the original set was constructed; in three cases the topology of the fold resembled that of the native protein. When the fold sets were filtered for the best scoring conformations with respect to an all-atom knowledge-based scoring function, the remaining subset of 50 structures yielded restraints of higher accuracy. A second round of distance geometry using these restraints resulted in an average coordinate RMS error of 4.38 A.     

Kinase inhibitors and the case for CH...O hydrogen bonds in protein-ligand binding

Although the hydrogen bond is known to be an important mediator of intermolecular interactions, there has yet to be an analysis of the role of CH...O hydrogen bonds in protein-ligand complexes. In this work, we present evidence for such nonstandard hydrogen bonds from a survey of aromatic ligands in 184 kinase crystal structures and 358 high-resolution structures from the Protein Data Bank. CH groups adjacent to the positively charged nitrogen of nicotinamide exhibit geometric preferences strongly suggestive of hydrogen bonding interactions, as do heterocyclic CH groups in kinase ligands, while other aromatic CH groups do not exhibit these characteristics. Ab initio calculations reveal a considerable range of CH...O hydrogen bonding potentials among different aromatic ring systems, with nicotinamide and heterocycles preferred in kinase inhibitors showing particularly favorable interactions. These results provide compelling evidence for the existence of CH...O hydrogen bonds in protein-ligand interactions, as well as information on the relative strength of various aromatic CH donors. Such knowledge will be of considerable value in protein modeling, ligand design, and structure-activity analysis.     

Hybrid alpha/beta3-peptides with proteinogenic side chains. Monosubstituted analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe.

The alpha/beta3-mixed tripeptides R-CO-beta3-HMet-Leu-Phe-OMe (1a,b), R-CO-Met-beta3-HLeu-Phe-OMe (2a,b) and R-CO-Met-Leu-beta3-HPhe-OMe (3a,b) (a, R = tert-butyloxy-; b, R = H-), analogues of the potent chemoattractant For-Met-Leu-Phe-OMe, have been synthesized by classical solution methods and fully characterized. The activities of the new analogues as chemoattractants, superoxide anion producers and lysozyme releasers have been determined on human neutrophils. Whereas all of the three N-formyl derivatives are significantly less active than the parent tripeptide as chemoattractants, compound 1b has been found to be highly active as a superoxide anion producer and 3b as a lysozyme releaser. The results show that the replacement of the native Leu residue at the central position is, in each of the examined cases, the least favourable modification. The three N-Boc derivatives are, as expected, devoid of activity as agonists, but they are all good inhibitors of chemotaxis. Information on the solution conformation has been obtained by examining the involvement of the NH groups in intramolecular H-bonds using 1H NMR. The conformation of the N-Boc analogue 1a has also been determined in the crystal state by x-ray diffraction analysis. The molecule is extended at the beta3-HMet residue (phi1 = -87 degrees; theta1 = 172 degrees; psi1 = 126 degrees) and no intramolecular H-bond is present.     

Use of structure comparison methods for the refinement of protein structure predictions. I. Identifying the structural family of a protein from low-resolution models.

Predicting the three-dimensional structure of proteins is still one of the most challenging problems in molecular biology. Despite its difficulty, several investigators have started to produce consistently low-resolution predictions for small proteins. However, in most of these cases, the prediction accuracy is still too low to make them useful. In the present article, we address the problem of obtaining better-quality predictions, starting from low-resolution models. To this end, we have devised a new procedure that uses these models, together with structure comparison methods, to identify the structural family of the target protein. This would allow, in a second step not described in the present work, to refine the predictions using conserved features of the identified family. In our approach, the structure database is investigated using predictions, at different accuracy levels, for a given protein. As query structures, we used both low-resolution versions of the native structures, as well as different sets of low accuracy predictions. In general, we found that for predictions with a resolution of > or =5-7 A, structure comparison methods were able to identify the fold of a protein in the top positions.     

Solvent effects on the conformational preferences of model peptoids. MP2 study

The influence of aqueous environment on the main‐chain conformation (ω₀, ?, and ψ dihedral angles) of two model peptoids: N‐acetyl‐N‐methylglycine N’‐methylamide (Ac‐N(Me)‐Gly‐NHMe) ( 1 ) and N‐acetyl‐N‐methylglycine N’,N’‐dimethylamide (Ac‐N(Me)‐Gly‐NMe₂) ( 2 ) was investigated by MP2/6‐311++G(d,p) method. The Ramachandran maps of both studied molecules with cis and trans configuration of the N‐terminal amide bond in the gas phase and in water environment were obtained and all energy minima localized. The polarizable continuum model was applied to estimate the solvation effect on conformation. Energy minima of the Ac‐N(Me)‐Gly‐NHMe and Ac‐N(Me)‐Gly‐NMe₂ have been analyzed in terms of the possible hydrogen bonds and C = O dipole attraction. To validate the theoretical results obtained, conformations of the similar structures gathered in the Cambridge Crystallographic Data Centre were analyzed. Obtained results indicate that aqueous environment in model peptoids 1 and 2 favors the conformation F (? and ψ = ?70º, 180º), and additionally significantly increases the percentage of structures with cis configuration of N‐terminal amide bond in studied compounds. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4.

Solid phase synthesis of BH4, the 26 amino-acid domain (6RTGYDNREIVMKYIHYKLSQRGYEWD31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant beta-sheeted structures in water that convert progressively into alpha-helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes.     

Solid-state IR-LD spectroscopic and theoretical analysis of glycine-containing peptides and their hydrochlorides

As part of an investigation on the coordination ability of peptides, structural analyses of the solid di-, tri, and tetrapeptides glycyl-glycine (GG), glycyl-glycyl-glycine (GGG), glycyl-glycyl-glycyl-glycine (GGGG), and their protonated hydrochlorides glycyl-glycine.HCl (GGH), glycyl-glycyl-glycine.HCl (GGGH), and glycyl-glycyl-glycyl-glycine.HCl (GGGGH) have been carried out. The quantum chemical calculations (Hartree-Fock/6-31++G**) and linear-dichroic infrared (IR-LD) spectroscopy predict a near to linear structure of the pure ligands, but the experimental IR-LD data are in accordance with a cross-linked disposition of amide fragments in the protonated forms.     

Amino acid deletion products resulting from incomplete deprotection of the Boc group from Npi-benzyloxymethylhistidine residues during solid-phase peptide synthesis.

In peptide synthesis, the use of N(alpha)-tert-butyloxycarbonyl-N(pi)-benzyloxymethylhistidine [Boc-His(pi-Bom)] raises the problem of the Bom group generating formaldehyde during the hydrogen fluoride (HF) cleavage reaction. This can lead to modification of the functional groups on amino acids in the peptide chain. Besides this side reaction, the failure of N(alpha)-Boc deprotection from the His(pi-Bom) residue occurs during TFA treatment for the standard solid-phase peptide synthesis (SPPS) even in the case of a non 'difficult sequence'. This gives amino acid deletion products generated at the N-terminus of the His(pi-Bom) residues. Reviewing the removability of the Boc group on amino acid derivatives showed that the group on the His(pi-Bom) residue was much more resistant under the deprotecting conditions than expected. To circumvent this problem, special precautions, i.e. prolonged deprotection steps and/or increased concentrations of TFA, should be taken for a successful SPPS.     

Ab Initio and DFT Study of the Structures,Vibrations and Energetics of the Urea Dimer and Trimer

Stable structures and electronic properties of small urea clusters are investigated with ab initio calculations. We optimized the cluster geometries and calculated the vibrational frequencies with Hartree–Fock (HF), second-order Møller-Plesset perturbation theory (MP2), and Density Functional Theory (DFT) methods using different basis sets. The most stable dimer was found to consist of two nonplanar urea molecules which are connected by two N–-H...O bonds in a common plane, and the most stable trimer has a flat structure of complex and planar C2 form for each urea molecule, like in the crystal. The interaction energies were corrected for the basis set superposition error (BSSE) using the full Boys*ndash;Bernardi counterpoise correction scheme. The stability of different dimer and trimer structures, the features of formation of H-bonds and presented here are compared to the available experimental data.     

Synthesis and conformational investigation of cyclic dipeptides: 7-membered rings containing alpha- and beta-amino acids.

The synthesis of heterocyclic compounds containing the 7-membered ring system [1,4]diazepane-2,5-dione is described. The aim of this study was to elaborate the solid phase and solution synthesis of eight representatives of the cyclic scaffold and to investigate their chemical stability and their conformational properties. The solid phase synthesis was performed on aminomethyl polystyrene resin using 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid as a backbone linker system (BAL-linker). After attachment of the alpha- and beta-amino acid and deprotection of the amino function, the dipeptide ester was obtained. The molecule was cyclized on the solid support by treatment with NaOMe in MeOH/NMP. The product was cleaved from the resin by TFA. For the solution pathway the linear dipeptides were synthesized by coupling of the BOC-protected L-alpha-amino acid with the beta2-amino acid ester (EDC/HOBT). After N- and C-terminal deprotection of the dipeptide, the linear species was cyclized with EDC/HOBT at a concentration of 3 mM in DMF. The products showed high chemical stability after storage in DMSO at room temperature for weeks. The x-ray and two dimensional NMR investigations were performed to investigate the conformation of the molecules. Three types of configuration could be distinguished by NMR, depending on the substitution pattern of the cyclic compounds. The x-ray results confirmed the NMR observations. In general the 7-membered rings showed rigidity, thus they could represent optimal scaffolds for new receptor ligands.     

Structure of the Escherichia coli ArnA N‐formyltransferase domain in complex with N5‐formyltetrahydrofolate and UDP‐Ara4N

ArnA from Escherichia coli is a key enzyme involved in the formation of 4‐amino‐4‐deoxy‐l ‐arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram‐negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Indeed, it is thought that such modifications may be responsible for the repeated infections of cystic fibrosis patients with Pseudomonas aeruginosa. ArnA is a bifunctional enzyme with the N‐ and C‐terminal domains catalyzing formylation and oxidative decarboxylation reactions, respectively. The catalytically competent cofactor for the formylation reaction is N¹⁰‐formyltetrahydrofolate. Here we describe the structure of the isolated N‐terminal domain of ArnA in complex with its UDP‐sugar substrate and N⁵‐formyltetrahydrofolate. The model presented herein may prove valuable in the development of new antimicrobial therapeutics.     

Enantioselective anion exchangers based on cinchona alkaloid‐derived carbamates: Influence of C8/C9 stereochemistry on chiral recognition

Four diastereomeric chiral stationary phases (CSPs) based on quinine, quinidine, epiquinine, and epiquinidine tert‐butyl carbamate selectors were synthesized and evaluated under ion exchange HPLC conditions with a set of racemic N‐acylated and N‐oxycarbonylated α‐amino acids as selectands. The enantioseparation potential of quinine‐ and quinidine‐derived CSPs proved to be far superior to that of their C₉‐epimeric congeners. The absolute configuration of C₉ stereogenic center of the cinchonan backbone of these selectors was identified as the structural feature controlling the elution order. Guided by an X‐ray structure of a most favorable selector–selectand complex and the observed chromatographic enantioseparation data, a chiral recognition model was advanced. The contributions of ion‐pairing, π–π donor–acceptor, hydrogen bonding and steric interactions were established as crucial factors. Chirality 11:522–528, 1999. © 1999 Wiley‐Liss, Inc.     

Properties and applications of the (2-nitrofluoren-9-yl)methoxycarbonyl group.

This paper presents a new protecting group, the (2-nitrofluoren-9-yl)methoxycarbonyl group. Investigations on the properties of this new modification of the Fmoc-system, such as the solvent-dependent photochemical cleavage, and enhanced lability towards bases, are described, as well as UV-kinetic measurements of the cleavage reaction. In addition, the incorporation of the (2-nitrofluoren-9-yl)methoxycarbonyl group into two peptides, and a sequence-dependent photochemical cleavage reaction are reported.     

Synthesis and conformational characterization of peptides related to the neck domain of a fungal kinesin.

The Y362K mutation in the neck domain of conventional kinesin from Neurospora crassa provokes a significant reduction of the rate of movement along microtubules. Since the alpha-helical coiled-coil structure of the neck region is implicated in the mechanism of the processive movement of kinesins, a series of peptides related to the heptad region 338-379 of the wild-type and the variant fungal kinesinswere synthesized as monomers and as N-terminal disulfide dimers, crosslinked to favour self-association into coiled-coil structures entropically. A comparison of the dichroic properties of the peptides and the effects of trifluoroethanol and peptide concentration clearly confirmed the strong implication of the single point mutation in destabilizing the intrinsic propensity of the peptides to fold into the supercoiled conformation. That there is a correlation between the stability of the coiled-coil and rate of movement of the kinesin is confirmed.     

Peptides containing 4-amino-1,2-dithiolane-4-carboxylic acid (Adt): conformation of Boc-Adt-Adt-NHMe and NH...S interactions.

To study the conformational preferences induced by the insertion of the 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) residue into a peptide backbone, the achiral N-protected dipeptide methylamide Boc-Adt-Adt-NHMe (1) was synthesized and its crystal state and solution conformation studied and compared with that exhibited by its carba-analogue Boc-Ac5c-Ac5c-NHMe containing two residues of 1-aminocyclopentane-1-carboxylic acid (Ac5c). Compound 1 in the crystal adopts a type-III beta-turn conformation and an analogous structure is that preferred in chloroform solution as established by 1H-NMR and NOE information. In the crystal packing three different Adt rings form a cavity and the involved sulphur atoms give rise to unusual multiple interactions with one NH group. The chemical nature of these intermolecular and intramolecular main-chain...side-chain NH...S interactions are discussed in terms of quantum chemical calculations.     

N-Terminal domain of HTLV-I integrase. Complexation and conformational studies of the zinc finger.

The HTLV-I integrase N-terminal domain [50-residue peptide (IN50)], and a 35-residue truncated peptide formed by residues 9-43 (IN35) have been synthesized by solid-phase peptide synthesis. Formation of the 50-residue zinc finger type structure through a HHCC motif has been proved by UV-visible absorption spectroscopy. Its stability was demonstrated by an original method using RP-HPLC. Similar experiments performed on the 35-residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50-residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV-I integrase N-terminal domain is likely to be composed of an helical region (residues 28-42) and a beta-strand (residues 20-23), associated with a HHCC zinc-binding motif. Size-exclusion chromatography showed that the structured zinc finger dimerizes through the helical region.