Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

High‐resolution crystal structures reveal a mixture of conformers of the Gly61‐Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus

Flavodoxins (Flds) are small proteins that shuttle electrons in a range of reactions in microorganisms. Flds contain a redox‐active cofactor, a flavin mononucleotide (FMN), and it is well established that when Flds are reduced by one electron, a peptide bond close to the FMN isoalloxazine ring flips to form a new hydrogen bond with the FMN N5H, stabilizing the one‐electron reduced state. Here, we present high‐resolution crystal structures of Flavodoxin 1 from Bacillus cereus in both the oxidized (ox) and one‐electron reduced (semiquinone, sq) state. We observe a mixture of conformers in the oxidized state; a 50:50 distribution between the established oxidized conformation where the peptide bond is pointing away from the flavin, and a conformation where the peptide bond is pointing toward the flavin, approximating the conformation in the semiquinone state. We use single‐crystal spectroscopy to demonstrate that the mixture of conformers is not caused by radiation damage to the crystal. This is the first time that such a mixture of conformers is reported in a wild‐type Fld. We therefore carried out a survey of published Fld structures, which show that several proteins have a pronounced conformational flexibility of this peptide bond. The degree of flexibility seems to be modulated by the presence, or absence, of stabilizing interactions between the peptide bond carbonyl and its surrounding amino acids. We hypothesize that the degree of conformational flexibility will affect the Fld ox/sq redox potential.     

A ligand-induced conformational change in the Yersinia protein tyrosine phosphatase.

Protein tyrosine phosphatases (PTPases) play critical roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. The structures of several different PTPases have revealed a conserved active site architecture in which a phosphate-binding loop, together with an invariant arginine, cradle the phosphate of a phosphotyrosine substrate and poise it for nucleophilic attack by an invariant cysteine nucleophile. We previously reported that binding of tungstate to the Yop51 PTPase from Yersinia induced a loop conformational change that moved aspartic acid 356 into the active site, where it can function as a general acid. This is consistent with the aspartic acid donating a proton to the tyrosyl leaving group during the initial hydrolysis step. In this report, using a similar structure of the inactive Cys 403-->Ser mutant of the Yersinia PTPase complexed with sulfate, we detail the structural and functional details of this conformational change. In response to oxyanion binding, small perturbations occur in active site residues, especially Arg 409, and trigger the loop to close. Interestingly, the peptide bond following Asp 356 has flipped to ligate a buried, active site water molecule that also hydrogen bonds to the bound sulfate anion and two invariant glutamines. Loop closure also significantly decreases the solvent accessibility of the bound oxyanion and could effectively shield catalytic intermediates from phosphate acceptors other than water. We speculate that the intrinsic loop flexibility of different PTPases may be related to their catalytic rate and may play a role in the wide range of activities observed within this enzyme family.     

Molecular dynamics simulations indicate that tyrosineB10 limits motions of distal histidine to regulate CO binding in soybean leghemoglobin

Myoglobin (Mb) uses strong electrostatic interaction in its distal heme pocket to regulate ligand binding. The mechanism of regulation of ligand binding in soybean leghemoglobin a (Lba) has been enigmatic and more so due to the absence of gaseous ligand bound atomic resolution three‐dimensional structure of the plant globin. While the 20‐fold higher oxygen affinity of Lba compared with Mb is required for its dual physiological function, the mechanism by which this high affinity is achieved is only emerging. Extensive mutational analysis combined with kinetic and CO‐FT‐IR spectroscopic investigation led to the hypothesis that Lba depended on weakened electrostatic interaction between distal HisE7 and bound ligand achieved by invoking B10Tyr, which itself hydrogen bonds with HisE7 thus restricting it in a single conformation detrimental to Mb‐like strong electrostatic interaction. Such theory has been re‐assessed here using CO‐Lba in silico model and molecular dynamics simulation. The investigation supports the presence of at least two major conformations of HisE7 in Lba brought about by imidazole ring flip, one of which makes hydrogen bonds effectively with B10Tyr affecting the former's ability to stabilize bound ligand, while the other does not. However, HisE7 in Lba has limited conformational freedom unlike high frequency of imidazole ring flips observed in Mb and in TyrB10Leu mutant of Lba. Thus, it appears that TyrB10 limits the conformational freedom of distal His in Lba, tuning down ligand dissociation rate constant by reducing the strength of hydrogen bonding to bound ligand, which the freedom of distal His of Mb allows. Proteins 2015; 83:1836–1848. © 2015 Wiley Periodicals, Inc.     

Transport of (Glyco)Sphingolipids in and Between Cellular Membranes; Multidrug Transporters and Lateral Domains

Sphingolipids are highly enriched in the outer leaflet of the plasma membrane lipid bilayer. However, the first glycolipid, glucosylceramide, is synthesized in the opposite, cytosolic leaflet of the Golgi membrane. This has led us to experiments which suggest that the level of glucosylceramide in the cytosolic surface is carefully regulated both by the balance between synthesis and hydrolysis and by transport away from this surface through translocators, multidrug transporters, the same molecules that make cancer cells resistant to chemotherapy. Our data suggest a role for newly synthesized glucosylceramide not only in the formation of domains in the luminal leaflet of the Golgi but also on the cytosolic surface of this organelle.     

Conformational analysis of invariant peptide sequences in bacterial genomes

The functional significance of evolutionarily conserved motifs/patterns of short regions in proteins is well documented. Although a large number of sequences are conserved, only a small fraction of these are invariant across several organisms. Here, we have examined the structural features of the functionally important peptide sequences, which have been found invariant across diverse bacterial genera. Ramachandran angles (phi,psi) have been used to analyze the conformation, folding patterns and geometrical location (buried/exposed) of these invariant peptides in different crystal structures harboring these sequences. The analysis indicates that the peptides preferred a single conformation in different protein structures, with the exception of only a few longer peptides that exhibited some conformational variability. In addition, it is noticed that the variability of conformation occurs mainly due to flipping of peptide units about the virtual C(alpha)...C(alpha) bond. However, for a given invariant peptide, the folding patterns are found to be similar in almost all the cases. Over and above, such peptides are found to be buried in the protein core. Thus, we can safely conclude that these invariant peptides are structurally important for the proteins, since they acquire unique structures across different proteins and can act as structural determinants (SD) of the proteins. The location of these SD peptides on the protein chain indicated that most of them are clustered towards the N-terminal and middle region of the protein with the C-terminal region exhibiting low preference. Another feature that emerges out of this study is that some of these SD peptides can also play the roles of "fold boundaries" or "hinge nucleus" in the protein structure. The study indicates that these SD peptides may act as chain-reversal signatures, guiding the proteins to adopt appropriate folds. In some cases the invariant signature peptides may also act as folding nuclei (FN) of the proteins.     

Mechanistic analyses of the suppression of amyloid β42 aggregation by apomorphine

(R)-Apomorphine (1) has the potential to reduce the accumulation of amyloid β-protein (Aβ42), a causative agent of Alzheimer’s disease (AD). Although the inhibition of Aβ42 aggregation by 1 is ascribable to the antioxidative effect of its phenol moiety, its inhibitory mechanism at the molecular level remains to be fully elucidated. LC–MS and UV analyses revealed that 1 is autoxidized during incubation to produce an unstable o-quinone form (2), which formed a Michael adduct with Lys 16 and 28 of Aβ42. A further autoxidized form of 1 (3) with o-quinone and phenanthrene moieties suppressed Aβ42 aggregation comparable to 1, whereas treating 1 with a reductant, tris(2-carboxyethyl)phosphine diminished its inhibitory activity. ¹H-¹⁵N SOFAST-HMQC NMR studies suggested that 1 interacts with Arg5, His13,14, Gln15, and Lys16 of the Aβ42 monomer. These regions form intermolecular β-sheets in Aβ42 aggregates. Since 3 did not perturb the chemical shift of monomeric Aβ42, we performed aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 to investigate whether 3 associates with Aβ42 oligomers. Compounds 1 and 3 delayed the onset of the oligomer-driven nucleation phase. Despite their cytotoxicity, they did not exacerbate Aβ42-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. These results demonstrate that extension of the conjugated system in 1 by autoxidation can promote its planarity, which is required for intercalation into the β-sheet of Aβ42 nuclei, thereby suppressing further aggregation.     

Capturing American White Ibises in urban South Florida using two novel techniques

As many wildlife species, including wading birds, adapt to anthropogenic landscapes and, in some cases, exhibit altered behaviors, studies that involve capturing birds may require new methods better suited for use in urban areas and to accommodate altered animal behavior. We developed two novel techniques, a leg lasso and flip net, for capturing American White Ibises (Eudocimus albus) in urban environments in southern Florida, and also used a traditional technique (mist‐nets) in non‐urban wetland habitats. The flip net and leg lasso were developed to capture White Ibises habituated to the presence of humans. Ibises were captured in urban and wetland environments from October 2015 to August 2017 in Palm Beach, Broward, and Lee counties, Florida. We captured 6.0 ± 13.5 ibis/h with the flip net, 1.6 ± 0.8 ibis/h with the leg lasso, and 0.5 ± 2.6 ibis/h with mist‐nets. We captured larger (higher mass to tarsus length ratio) birds using the flip net and leg lasso than using mist‐nets, and captured more males with leg lassos than with other two techniques. The novel techniques we used are efficient, cost effective, easy to use, and also potentially useful for capturing other species of birds. Leg lassos and flip nets are also safe to use in populated areas for both birds and humans.     

Molecular dynamics simulation to investigate anhydrous phosphoric acid-doped polybenzimidazole

The study conducted a molecular dynamics simulation based on a condensed-phase optimised molecular potentials for atomistic simulation studies force field model to investigate an anhydrous system of phosphoric acid-doped polybenzimidazole (poly[2,2′-(m-phenylene)-5,5′-bibenzimidazole], PBI). Intermolecular pair correlation functions and corresponding coordination numbers were calculated to research the strengths for various types of hydrogen bonding. The results display that the strengths of the hydrogen bonding interactions are in the order of o1–h pair > o2–h pair > n2a–h pair > n3a–h pair, and most protons are located around the neighbourhood of H₂PO₄^– rather than that of PBI. The proton conductivities are 3.86 × 10^?3 S cm^?1 at 298 K and 8.50 × 10^?3 S cm^?1 at 413 K. Moreover, the value obtained from our simulation system at 413 K is within the same order of magnitude as the experimentally measured value 0.012 S cm^?1 at 420% doping level. The distribution of proton displacement exhibits that the displacement of most protons is about 1.25–2.5 Å. The displacement is over 3.0 Å only for a fraction of protons. In addition, the greatest displacement can approach 4.595 Å. The trajectory analyses of protons show that the most possible mechanisms of proton transfer come from three ways: (a) between two H₂PO₄^– anions, (b) between H₂PO₄^– anions and benzimidazole moieties and (c) between two benzimidazole moieties. The dynamics of polymer motion was studied by the trajectory analyses of ring flips. The large amplitude flips of rings in the polymer chains were found in the system. The flips between benzene and benzimidazole are more frequent than that between benzimidazole moieties.     

Simulation of the β- to α-sheet transition results in a twisted sheet for antiparallel and an α-nanotube for parallel strands: implications for amyloid formation

α-sheet has been proposed to be the main constituent of the toxic amyloid intermediate. Molecular dynamics simulations on proteins known to be involved in amyloid diseases have demonstrated that β-sheet can, under certain conditions, spontaneously convert to α-sheet via ββ→α(R)α(L) peptide-plane flipping. Using torsion-angle driving to simulate this flip the transition has been investigated for parallel and antiparallel sheets. Concerted and sequential flipping processes were simulated, the former allowing direct calculation of helical parameters. For antiparallel sheet, the strands tend to splay apart during the transition. This can be understood by consideration of the geometry of repeating dipeptide conformations. At the end of the transition antiparallel α-sheet is slightly twisted, comprising gently curving strands. In parallel sheet, the strands maintain identical conformations and stay hydrogen bonded during the transition as they curl up to suggest a hitherto unseen structure, the multi-helix α-nanotube. Intriguingly, the α-nanotube has some of the characteristics of the parallel β-helix, a single-helix structure also implicated in amyloid. Unlike the β-helix, α-nanotube formation could involve identical strands aligning with each other in register as in most amyloids.