Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

The Strait of Gibraltar as a major biogeographic barrier in Mediterranean conifers: a comparative phylogeographic survey

The Strait of Gibraltar (SG) is reputed for being both a bridge and a geographic barrier to biological exchanges between Europe and Africa. Major genetic breaks associated with this strait have been identified in various taxa, but it is unknown whether these disjunctions have been produced simultaneously or by independent biogeographic processes. Here, the genetic structure of five conifers distributed on both sides of the SG was investigated using mitochondrial (nad1 b/c, nad5-1, nad5-4 and nad7-1) and chloroplast (Pt1254, Pt15169, Pt30204, Pt36480, Pt71936 and Pt87268) DNA markers. The distribution of genetic variation was partially congruent between types of markers within the same species. Across taxa, there was a significant overlapping between the SG and the genetic breaks detected, especially for the four Tertiary species surveyed (Abies pinsapo complex, Pinus nigra, Pinus pinaster and Taxus baccata). For most of these taxa, the divergence of populations across the SG could date back to long before the Pleistocene glaciations. However, their strongly different cpDNA G(ST) and R(ST) values point out that they have had dissimilar population histories, which might include contrasting amounts of pollen-driven gene flow since their initial establishment in the region. The fifth species, Pinus halepensis, was genetically depauperated and homogenous on both sides of the SG. A further analysis of nuclear DNA sequences with coalescent-based isolation with migration models suggests a Pleistocene divergence of P. halepensis populations across the SG, which is in sharp contrast with the pre-Pleistocene divergence dates obtained for P. pinaster. Altogether, these results indicate that the genetic breaks observed across this putative biogeographical barrier have been produced by independent evolutionary processes related to the biological history of each individual species instead of a common vicariant phenomenon.     

Distinct Components of Retrograde CaV1.1-RyR1 Coupling Revealed by a Lethal Mutation in RyR1

The molecular basis for excitation-contraction coupling in skeletal muscle is generally thought to involve conformational coupling between the L-type voltage-gated Ca²⁺ channel (Ca_V1.1) and the type 1 ryanodine receptor (RyR1). This coupling is bidirectional; in addition to the orthograde signal from Ca_V1.1 to RyR1 that triggers Ca²⁺ release from the sarcoplasmic reticulum, retrograde signaling from RyR1 to Ca_V1.1 results in increased amplitude and slowed activation kinetics of macroscopic L-type Ca²⁺ current. Orthograde coupling was previously shown to be ablated by a glycine for glutamate substitution at RyR1 position 4242. In this study, we investigated whether the RyR1-E4242G mutation affects retrograde coupling. L-type current in myotubes homozygous for RyR1-E4242G was substantially reduced in amplitude (∼80%) relative to that observed in myotubes from normal control (wild-type and/or heterozygous) myotubes. Analysis of intramembrane gating charge movements and ionic tail current amplitudes indicated that the reduction in current amplitude during step depolarizations was a consequence of both decreased Ca_V1.1 membrane expression (∼50%) and reduced channel P_o (∼55%). In contrast, activation kinetics of the L-type current in RyR1-E4242G myotubes resembled those of normal myotubes, unlike dyspedic (RyR1 null) myotubes in which the L-type currents have markedly accelerated activation kinetics. Exogenous expression of wild-type RyR1 partially restored L-type current density. From these observations, we conclude that mutating residue E4242 affects RyR1 structures critical for retrograde communication with Ca_V1.1. Moreover, we propose that retrograde coupling has two distinct and separable components that are dependent on different structural elements of RyR1.     

Individualities in a flock of free-roaming greylag geese: behavioral and physiological consistency over time and across situations

The concept of personality implies individual differences in behavior and physiology that show some degree of repeatability/consistency over time and across contexts. Most studies of animal personality, particularly studies of individuals' variation in physiological mechanisms, have been conducted on selected individuals in controlled conditions. We attempted to detect consistent behaviors as well as physiological patterns in greylag ganders (Anser anser) from a free-roaming flock living in semi-natural conditions. We tested 10 individuals repeatedly, in a handling trial, resembling tests for characterization of "temperaments" in captive animals. We recorded the behavior of the same 10 individuals during four situations in the socially intact flock: (1) a "low density feeding condition", (2) a "high density feeding condition", (3) a "low density post-feeding situation" and (4) while the geese rested. We collected fecal samples for determination of excreted immuno-reactive corticosterone (BM) and testosterone metabolites (TM) after handling trials, as well as the "low density feeding" and the "high density feeding" conditions. BM levels were very highly consistent over the repeats of handling trials, and the "low density feeding condition" and tended to be consistent over the first two repeats of the "high density feeding condition". Also, BM responses tended to be consistent across contexts. Despite seasonal variation, there tended to be inter-test consistency of TM, which pointed to some individual differences in TM as well. Aggressiveness turned out to be a highly repeatable trait, which was consistent across social situations, and tended to correlate with an individual's resistance during handling trials. Also, "proximity to the female partner" and "sociability" - the average number of neighboring geese in a close distance while resting - were consistent. We conclude that aggressiveness, "affiliative tendencies" and levels of excreted corticosterone and testosterone metabolites may be crucial factors of personality in geese.     

Intracellular Localization of Poliovirus Plus- and Minus-Strand RNA Visualized by Strand-Specific Fluorescent In Situ Hybridization

The time courses of poliovirus plus- and minus-strand RNA synthesis in infected HEp-2 cells were monitored separately, using a quantitative RNase assay. In parallel, viral RNA and proteins were located in situ by confocal microscopy within cells fixed by a protocol determined to retain their native size and shape. Plus- and minus-strand RNAs were visualized by fluorescent in situ hybridization (FISH) with strand-specific riboprobes. The probes were labelled with different fluorochromes to allow for the simultaneous detection of plus- and minus-strand RNA. The FISH experiments showed minus-strand RNA to be present in distinct, regularly sized, round structures throughout the viral replication cycle. Plus-strand RNA was found in the same structures and also in smaller clusters of vesicles. Association of viral RNA with membranes was demonstrated by combining FISH with immunofluorescence (IF) detection of the viral 2B- and 2C-containing P2 proteins, which are known to be markers for virus-induced membranes. At early times postinfection, the virus-induced membranous structures were distributed through most of the cytoplasm, whereas around peak RNA synthesis, both RNA-associated membranous structures migrated to the center of the cell. During this process, the plus- and minus-strand-containing larger structures stayed as recognizable entities, whereas the plus-strand-containing granules coalesced into a juxtanuclear area of membranous vesicles. An involvement of Golgi-derived membranes in the formation of virus-induced vesicles and RNA synthesis early in infection was investigated by IF with 2C- and Golgi-specific antibodies.     

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

Fermentanomics informed amino acid supplementation of an antibody producing mammalian cell culture

Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools. Published 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:745–753, 2013