Amphipathic motifs in BAR domains are essential for membrane curvature sensing

BAR (Bin/Amphiphysin/Rvs) domains and amphipathic α‐helices (AHs) are believed to be sensors of membrane curvature thus facilitating the assembly of protein complexes on curved membranes. Here, we used quantitative fluorescence microscopy to compare the binding of both motifs on single nanosized liposomes of different diameters and therefore membrane curvature. Characterization of members of the three BAR domain families showed surprisingly that the crescent‐shaped BAR dimer with its positively charged concave face is not able to sense membrane curvature. Mutagenesis on BAR domains showed that membrane curvature sensing critically depends on the N‐terminal AH and furthermore that BAR domains sense membrane curvature through hydrophobic insertion in lipid packing defects and not through electrostatics. Consequently, amphipathic motifs, such as AHs, that are often associated with BAR domains emerge as an important means for a protein to sense membrane curvature. Measurements on single liposomes allowed us to document heterogeneous binding behaviour within the ensemble and quantify the influence of liposome polydispersity on bulk membrane curvature sensing experiments. The latter results suggest that bulk liposome‐binding experiments should be interpreted with great caution.     

A Frustrated Binding Interface for Intrinsically Disordered Proteins

Intrinsically disordered proteins are very common in the eukaryotic proteome, and many of them are associated with diseases. Disordered proteins usually undergo a coupled binding and folding reaction and often interact with many different binding partners. Using double mutant cycles, we mapped the energy landscape of the binding interface for two interacting disordered domains and found it to be largely suboptimal in terms of interaction free energies, despite relatively high affinity. These data depict a frustrated energy landscape for interactions involving intrinsically disordered proteins, which is likely a result of their functional promiscuity.     

Product profiles in enzymic and non-enzymic oxidations of the lignin model compound erythro-1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol

The erythro form of the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (1) was oxidized with laccase/ABTS, lead(IV) tetraacetate (LTA), lignin peroxidase/H₂O₂, cerium(IV) ammonium nitrate (CAN) and Fenton's reagent. The product profiles obtained with the different oxidants were compared after separation, identification and quantification of the products using HPLC, UV-diode array detector and electrospray ionization mass spectrometry in positive ionization mode. The oxidants generated different product profiles that reflected their different properties. Oxidation with laccase/ABTS resulted almost exclusively in formation of 1-(3,4-dimethoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)-1-propanone (2). Oxidation with LTA resulted in more 3,4-dimethoxybenzaldehyde (3) than ketone 2. Lignin peroxidase and CAN gave similar product profiles and aldehyde 3 was the predominant product (only small amounts of ketone 2 were formed). Oxidation with Fenton's reagent resulted in the formation of more aldehyde 3 than ketone 2 but the yields were very low. CAN served as an excellent model for the lignin peroxidase-catalyzed oxidation, while the laccase-mediator system, LTA and Fenton's reagent provided distinctly different product profiles. Erythro-1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol was present among the products obtained on oxidation with LTA, lignin peroxidase, CAN and Fenton's reagent. The differences in redox potential between the oxidants afford an explanation of the diverse product patterns but other factors may also be of importance. The reactions leading to cleavage of the β-ether bond with formation of 1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol (veratrylglycerol) were found to proceed without affecting the configuration at the β-carbon atom.     

Cytological and morphological variation in Bellevalia

Material from 45 populations representing 15 species of Bellevalia were analyzed as to chromosome numbers and morphology. For 3 species reports are new, viz. for B. clusiana (2x), B, tauri (2x, 4x), and B. nivalis (4x). In B. saviczii diploids, tetraploids and hexaploids were found. The basic Bellevalia karyotype prevails, but chromosome heteromorphism is rather common at the populational level. B–chromosomes were found in some populations of B. glauca s.l. and B. saviczii. Discussions on karyotype variation, polyploidy, morphologic variation and generic delimitation are presented.     

Specificity modulation of barley alpha-amylase through biased random mutagenesis involving a conserved tripeptide in beta --> alpha loop 7 of the catalytic (beta/alpha)(8)-barrel domain.

The relative specificity and bond cleavage pattern of barley alpha-amylase 1 (AMY1) were dramatically changed by mutation in F(286)VD that connected beta-strand 7 of the catalytic (beta/alpha)(8)-barrel to a succeeding 3(10)-helix. This conserved tripeptide of the otherwise variable beta --> alpha segment 7 lacked direct ligand contact, but the nearby residues His290 and Asp291 participated in transition-state stabilization and catalysis. On the basis of sequences of glycoside hydrolase family 13, a biased random mutagenesis protocol was designed which encoded 174 putative F(286)VD variants of C95A-AMY1, chosen as the parent enzyme to avoid inactivating glutathionylation by the yeast host. The FVG, FGG, YVD, LLD, and FLE mutants showed 12-380 and 1.8-33% catalytic efficiency (k(cat)/K(m)) toward 2-chloro-4-nitrophenyl beta-D-maltoheptaoside and amylose DP17, respectively, and 0.5-50% activity for insoluble starch compared to that of C95A-AMY1. K(m) and k(cat) were decreased 2-9- and 1.3-83-fold, respectively, for the soluble substrates. The starch:oligosaccharide and amylose:oligosaccharide specificity ratios were 13-172 and 2.4-14 for mutants and 520 and 27 for C95A-AMY1, respectively. The FVG mutant released 4-nitrophenyl alpha-D-maltotrioside (PNPG(3)) from PNPG(5), whereas C95A-AMY1 produced PNPG and PNPG(2). The mutation thus favored interaction with the substrate aglycon part, while products from PNPG(6) reflected the fact that the mutation restored binding at subsite -6 which was lost in C95A-AMY1. The outcome of this combined irrational and rational protein engineering approach was evaluated considering structural accommodation of mutant side chains. FVG and FGG, present in the most active variants, represented novel sequences. This emphasized the worth of random mutagenesis and launched flexibility as a goal for beta --> alpha loop 7 engineering in family 13.     

Crystallization and preliminary X-ray diffraction studies of a peroxidase from barley grain.

Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P2(1)2(1)2(1) with unit cell dimensions a = 62.95 A, b = 66.24 A and c = 70.78 A. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 A.     

High Female Mortality Resulting in Herd Collapse in Free-Ranging Domesticated Reindeer (Rangifer tarandus tarandus) in Sweden

Reindeer herding in Sweden is a form of pastoralism practised by the indigenous Sámi population. The economy is mainly based on meat production. Herd size is generally regulated by harvest in order not to overuse grazing ranges and keep a productive herd. Nonetheless, herd growth and room for harvest is currently small in many areas. Negative herd growth and low harvest rate were observed in one of two herds in a reindeer herding community in Central Sweden. The herds (A and B) used the same ranges from April until the autumn gathering in October–December, but were separated on different ranges over winter. Analyses of capture-recapture for 723 adult female reindeer over five years (2007–2012) revealed high annual losses (7.1% and 18.4%, for herd A and B respectively). A continuing decline in the total reindeer number in herd B demonstrated an inability to maintain the herd size in spite of a very small harvest. An estimated breakpoint for when herd size cannot be kept stable confirmed that the observed female mortality rate in herd B represented a state of herd collapse. Lower calving success in herd B compared to A indicated differences in winter foraging conditions. However, we found only minor differences in animal body condition between the herds in autumn. We found no evidence that a lower autumn body mass generally increased the risk for a female of dying from one autumn to the next. We conclude that the prime driver of the on-going collapse of herd B is not high animal density or poor body condition. Accidents or disease seem unlikely as major causes of mortality. Predation, primarily by lynx and wolverine, appears to be the most plausible reason for the high female mortality and state of collapse in the studied reindeer herding community.     

Occurrence and chirality of oscillaxanthin

The quantitative carotenoid composition of natural blooms of Oscillatoria rubescens and O. agardhii is reported and compared with previous isolations. Chemical or enzymatic conversion of oscillaxanthin to the chiral aglycone failed. CD-correlation of oscillaxanthin hexaacetate with (2S,2′S)-bacterioruberin, (2′R)-plectaniaxanthin and (2′R)-plectaniaxanthin-2′-β-D-glucoside tetraacetate support the 2R,2′R-configuration for oscillaxanthin.     

Selectivity of the surface binding site (SBS) on barley starch synthase I

Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity for malto-triose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β-cyclodextrin (β-CD) albeit with a measurable K _D for only β-CD and M7. Moreover, an HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. This behaviour suggests that a chain in the α-glucan molecule (amylopectin) that is undergoing extension attaches itself at the SBS and that the active site itself, likely working on a different end chain, has low affinity for both substrate and product.