Toward engineering efficient peptidomimetics. Screening conformational landscape of two modified dehydroaminoacids

Effective peptidomimetics should posses structural rigidity and appropriate interaction pattern leading to potential spatial and electronic matching to the target receptor site. Rational design of such small bioactive molecules could push chemical synthesis and molecular modeling toward faster progress in medicinal chemistry. Conformational properties of N‐t‐butoxycarbonyl‐glycine‐(E/Z)‐dehydrophenylalanine N′,N′‐dimethylamides (Boc‐Gly‐(E/Z)‐ΔPhe‐NMe₂) in chloroform were studied by NMR and IR spectroscopy. The experimental findings were supported by extensive calculations at DFT(B3LYP, M06‐2X) and MP2 levels of theory and the β‐turn tendency for both isomers of the studied dipeptide were determined in vacuum and in solution. The theoretical data and experimental IR results were used as an additional information for the NMR‐based determination of the detailed solution conformations of the peptides. The obtained results reveal that N‐methylation of C‐terminal amide group changes dramatically the conformational properties of studied dehydropeptides. Theoretical conformational analysis reveals that the tendency to adopt β‐turn conformations is much weaker for the N‐methylated Z isomer (Boc‐Gly‐(Z)‐ΔPhe‐NMe₂), both in vacuum and in polar environment. On the contrary, N‐methylated E isomer (Boc‐Gly‐(E)‐ΔPhe‐NMe₂) can easily adopt β‐turn conformation, but the backbone torsion angles (φ₁, ψ₁, φ₂, ψ₂) are off the limits for common β‐turn types. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 28–40, 2014.     

Molecular phylogeography of Carapichea ipecacuanha,an amphitropical shrub that occurs in the understory of both semideciduous and evergreen forests

The medicinal shrub Carapichea ipecacuanha (ipecac) is an amphitropic species with three disjunct areas of distribution. In the Brazilian Atlantic and Amazonian ranges, the species was associated mostly with the understory of seasonal semideciduous forests, whereas in the Central American–Colombian range, the species occurred in the understory of moist evergreen forests. We examined the phylogeographic structure of ipecac using chloroplast trnT‐trnL and nuclear internal transcribed spacer (ITS) sequences from 120 and 46 specimens, respectively. To complement existing data on root alkaloid profiles, we used high‐performance liquid chromatography to assess the levels of emetine and cephaeline in 33 specimens from the two Brazilian ranges. The three ranges shared neither nuclear nor chloroplast haplotypes. The phylogeographic structures showed an uneven distribution of genetic diversity, sharp breaks and high levels of genetic differentiation among ranges. Our results suggest that the extant populations are descendents of at least four distinct ancestral lineages. The Atlantic ipecacs showed higher levels of genetic diversity than ipecacs from the other two ranges; it is likely that they derive from two ancestral lineages, with long‐term persistence in that region. The Amazonian ipecacs were monomorphic with respect to the ITS and cpDNA sequences, which supports the view that there was a recent expansion from a single parental source after a strong genetic bottleneck. The existence of a fourth distinct lineage is apparent from the high levels of genetic and chemical differentiation that we identified in the Central American–Columbian ipecacs.     

Cosolvent-assisted oxidative folding of a bicyclic alpha-conotoxin ImI.

alpha-Conotoxin ImI is a 12-amino acid peptide, found in the venom of the marine snail Conus imperialis. This conotoxin is a selective antagonist of alpha7 nicotinic acetylcholine receptors. To produce biologically active alpha-ImI, disulfide bonds must be formed between Cys2-Cys8 and Cys3-Cys12. Oxidative folding of bicyclic conotoxins, such as alpha-ImI, has been traditionally achieved using two-step oxidation protocols with orthogonal protection on two native pairs of cysteines. In this work, two alternative oxidation protocols were explored: (1) the recently described one-pot oxidation of t-butyl/4-methylbenzyl protected Cys pairs and (2) direct oxidative folding. In contrast to the first method, the latter one resulted in high yields of correctly folded alpha-ImI. The addition of organic cosolvents, such as methanol, ethanol or isopropanol into the folding mixture significantly increased the accumulation of the native peptide. This effect was also observed for another conotoxin, alpha-PnIA. It is suggested that cosolvent-assisted direct oxidation might be of general use for other bicyclic alpha-conotoxins, but efficiency should be assessed on a case-by-case basis.     

Extrapolation of water and formaldehyde harmonic and anharmonic frequencies to the B3LYP/CBS limit using polarization consistent basis sets

The harmonic and anharmonic frequencies of fundamental vibrations in formaldehyde and water were successfully estimated using the B3LYP Kohn-Sham limit. The results obtained with polarization- and correlation-consistent basis sets were fitted with a two-parameter formula. Anharmonic corrections were obtained by a second order perturbation treatment (PT2). We compared the performance of the PT2 scheme on the two title molecules using SCF, MP2 and DFT (BLYP, B3LYP, PBE and B3PW91 functionals) methods combined with polarization consistent pc-n (n = 0, 1, 2, 3, 4) basis sets, Dunning’s basis sets (aug)-cc-pVXZ where X = D, T, Q, 5, 6 and Pople’s basis sets up to 6-311++G(3df,2pd). The influence of SCF convergence level and density grid size on the root mean square of harmonic and anharmonic frequency deviations from experimental values was tested. The wavenumber of formaldehyde CH₂ anharmonic asymmetric stretching mode is very sensitive to grid size for large basis sets; this effect is not observed for harmonic modes. BLYP-calculated anharmonic frequencies consistently underestimate observed wavenumbers. On the basis of formaldehyde anharmonic frequencies, we show that increasing the Pople basis set size does not always lead to improved agreement between anharmonic frequencies and experimental values.     

Effects of grazing on plant species diversity and carbon partitioning in semiarid rangelands of northeastern China

Grasslands are one of the most widespread landscapes worldwide, covering approximately one-fifth of the world’s land surface, where grazing is a common practice. How carbon storage responds to grazing in steppes remains poorly understood. We quantified the effects of grazing on community composition and species diversity, and carbon storage in two typical grasslands of northeastern China, one in Horqin and the other one in Hulunbeier. In both grasslands, grazing did not influence plant species diversity. However, it substantially decreased aboveground carbon by 31% and 54% in Horqin and Hulunbeier, respectively. Fenced and grazing treatments showed a similar belowground carbon at both locations. The predominant carbon pool in the study grassland ecosystem was found in the upper 100 cm soil depth, from 98.2 to 99.1% of the total carbon storage. There were no significant effects of grazing on soil carbon neither in the whole profile nor in the uppermost 20 cm soil depth in the two study grasslands. Studies on the effects of varying rangeland management, such as region disparity and grazing systems, may have important consequences on species diversity and carbon partitioning, and thus on rangeland stability and ecosystem functioning.     

Seed characteristics and fatty acid composition of castor (<i>Ricinus communis</i> L.) varieties in Northeast China

Oil content and fatty acid composition were investigated on 12 castor varieties and strains by using the soxhlet extraction method and capillary gas chromatography. This was made to provide a reference and theoretical basis for castorbean breeding with high oil content, determine variability of seed compounds for breeding purposes, and broaden chemical material choices. Results revealed that crude fat percentage in seeds ranged from 18.91 to 35.84% with an average of 25.91%; the absolute content of ricinoleic acid varied between 171.65 g/kg and 314.03 g/kg with an average of 222.43 g/kg, and kernel crude fat percentage was between 24.28 and 46.97% with an average of 34.30%. All these study variables were highest in the 2129 strain. The percentage of ricinoleic acid in crude fat was between 83.85 to 87.62%, and the highest value was found in the zhebi4 accession. The other fatty acids appeared in small concentrations, and showed small amplitude: 1.12 to 1.61%, 1.21 to 1.61%, 3.53 to 4.80%, 5.35 to 6.38%, 0.52 to 0.79%, 0.05 to 0.08% and 0.43 to 0.55%, for palmitic, stearic, oleic, linolic, linolenic, arachidic, and arachidonic acids, respectively. One hundred seed weight was determined for each accession. One hundred seed weight ranged from 25.7 g to 34.0 g with an average of 29.9 g. There was a significant correlation between seed weight and oil content, but the correlation value was low (r=0.51). Cluster analysis by SSPS based on the content of fatty acid composition revealed that the accessions were divided into three independent clusters. These findings will clearly provide useful information for further research in breeding and utilization of castor oil.     

Analysis of serine proteinase-inhibitor interaction by alanine shaving

We analyzed the energetic importance of residues surrounding the hot spot (the P(1) position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving. One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s). Although truncation of the side chains of residues surrounding the P(1) position to methyl groups caused a decrease in Delta G(den) values up to 6.4 kcal mole(-1), it did not influence the overall conformation of the inhibitor. We found that the replacement of up to six residues with alanines was fully additive at the level of protein stability. To analyze the influence of the structural epitope on the association energy, we determined association constants for BPTI variants and both enzymes and applied the additivity analysis. Shaving of two binding loops led to a progressive drop in the association energy, more pronounced for trypsin (decrease up to 9.6 kcal mole(-1)) than chymotrypsin (decrease up to 3.5 kcal mole(-1)). In the case of extensively mutated variants interacting with chymotrypsin, the association energies agreed very well with the values calculated from single mutational effects. However, when P(1)-neighboring residues were shaved to alanine(s), their contribution to the association energy was not fully removed because of the presence of methyl groups and main chain-main chain intermolecular hydrogen bonds. Moreover, the hot spot had a different contribution to the complex stability in the fully shaved BPTI variant compared with the wild type, which was caused by perturbations of the P(1)-S(1) electrostatic interaction.     

Structure and sodium channel activity of an excitatory I1-superfamily conotoxin

Conotoxin iota-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I1-superfamily, which contains eight cysteine residues arranged in a -C-C-CC-CC-C-C- pattern. iota-RXIA (formerly designated r11a) is one of three characterized I1 peptides in which the third last residue is posttranslationally isomerized to the d configuration. Naturally occurring iota-RXIA with d-Phe44 is significantly more active as an excitotoxin than the l-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I1-superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that iota-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the d-Phe44 and l-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of iota-RXIA as a voltage-gated sodium channel; iota-RXIA is an agonist, shifting the voltage dependence of activation of mouse NaV1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in iota-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.