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1.	Alm Rosenblad, Magnus, 1957, et al. (författare) Detection of signal recognition particle (SRP) RNAs in the nuclear ribosomal internal transcribed spacer 1 (ITS1) of three lineages of ectomycorrhizal fungi (Agaricomycetes, Basidiomycota) 2016 Ingår i: MycoKeys. - : Pensoft Publishers. - 1314-4057 .- 1314-4049. ; 13, s. 21-33 Tidskriftsartikel (refereegranskat)abstract During a routine scan for Signal Recognition Particle (SRP) RNAs in eukaryotic sequences, we surprisingly found in silico evidence in GenBank for a 265-base long SRP RNA sequence in the ITS1 region of a total of 11 fully identified species in three ectomycorrhizal genera of the Basidiomycota (Fungi): Astraeus, Russula, and Lactarius. To rule out sequence artifacts, one specimen from a species indicated to have the SRP RNA-containing ITS region in each of these genera was ordered and re-sequenced. Sequences identical to the corresponding GenBank entries were recovered, or in the case of a non-original but conspecific specimen differed by three bases, showing that these species indeed have an SRP RNA sequence incorporated into their ITS1 region. Other than the ribosomal genes, this is the first known case of non-coding RNAs in the eukaryotic ITS region, and it may assist in the examination of other types of insertions in fungal genomes.
2.	Trona, Federica, et al. (författare) Neural coding merges sex and habitat chemosensory signals in an insect herbivore 2013 Ingår i: Proceedings of the Royal Society of London. Biological Sciences. - : The Royal Society. - 0962-8452 .- 1471-2954. ; 280:1760, s. 20130267- Tidskriftsartikel (refereegranskat)abstract Understanding the processing of odour mixtures is a focus in olfaction research. Through a neuroethological approach, we demonstrate that different odour types, sex and habitat cues are coded together in an insect herbivore. Stronger flight attraction of codling moth males, Cydia pomonella, to blends of female sex pheromone and plant odour, compared with single compounds, was corroborated by functional imaging of the olfactory centres in the insect brain, the antennal lobes (ALs). The macroglomerular complex (MGC) in the AL, which is dedicated to pheromone perception, showed an enhanced response to blends of pheromone and plant signals, whereas the response in glomeruli surrounding the MGC was suppressed. Intracellular recordings from AL projection neurons that transmit odour information to higher brain centres, confirmed this synergistic interaction in the MGC. These findings underscore that, in nature, sex pheromone and plant odours are perceived as an ensemble. That mating and habitat cues are coded as blends in the MGC of the AL highlights the dual role of plant signals in habitat selection and in premating sexual communication. It suggests that the MGC is a common target for sexual and natural selection in moths, facilitating ecological speciation.
3.	Ding, Baojian, et al. (författare) Sequence variation determining stereochemistry of a delta-11 desaturase active in moth sex pheromone biosynthesis 2016 Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 1879-0240 .- 0965-1748. ; 74, s. 68-75 Tidskriftsartikel (refereegranskat)abstract A Δ11 desaturase from the oblique banded leaf roller moth Choristoneura rosaceana takes the saturated myristic acid and produces a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer (35:65). A desaturase from the spotted fireworm moth Choristoneura parallela also operates on myristic acid substrate but produces almost pure (E)-11-tetradecenoate. The two desaturases share 92% amino acid identity and 97% amino acid similarity. There are 24 amino acids differing between these two desaturases. We constructed mutations at all of these positions to pinpoint the sites that determine the product stereochemistry. We demonstrated with a yeast functional assay that one amino acid at the cytosolic carboxyl terminus of the protein (258E) is critical for the Z activity of the C. rosaceana desaturase. Mutating the glutamic acid (E) into aspartic acid (D) transforms the C. rosaceana enzyme into a desaturase with C. parallela-like activity, whereas the reciprocal mutation of the C. parallela desaturase transformed it into an enzyme producing an intermediate 64:36 E/Z product ratio. We discuss the causal link between this amino acid change and the stereochemical properties of the desaturase and the role of desaturase mutations in pheromone evolution.
4.	Mamontov, Eugen, 1955, et al. (författare) Mathematical models and numerical simulation results for oncogeny: Specific problems and different tools for various user communities 2008 Ingår i: Abstracts Booklet. CancerSim2008 — Euroconference on Modelling and Simulation of Cancer Growth and Therapy, 19-21 May, Turin, Italy; N. Bellomo and G. Forni (Chairmen) http://www.cancersim2008.org. Konferensbidrag (refereegranskat)
5.	Tavella, T A, et al. (författare) Yeast-based high-throughput screens for discovery of kinase inhibitors for neglected diseases. 2021 Ingår i: Advances in Protein Chemistry and Structural Biology. - : Elsevier. - 1876-1631. ; 124, s. 275-309 Forskningsöversikt (refereegranskat)abstract The discovery and development of a new drug is a complex, time consuming and costly process that typically takes over 10 years and costs around 1 billion dollars from bench to market. This scenario makes the discovery of novel drugs targeting neglected tropical diseases (NTDs), which afflict in particular people in low-income countries, prohibitive. Despite the intensive use of High-Throughput Screening (HTS) in the past decades, the speed with which new drugs come to the market has remained constant, generating doubts about the efficacy of this approach. Here we review a few of the yeast-based high-throughput approaches that can work synergistically with parasite-based, in vitro, or in silico methods to identify and optimize novel antiparasitic compounds. These yeast-based methods range from HTP screens to identify novel hits against promising parasite kinase targets to the identification of potential antiparasitic kinase inhibitors extracted from databases of yeast chemical genetic screens.
6.	Nachin, Laurence, 1971, et al. (författare) Heterodimer formation within universal stress protein classes revealed by an in silico and experimental approach. 2008 Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 380:2, s. 340-50 Tidskriftsartikel (refereegranskat)abstract Universal stress proteins (Usps) are found in all kingdoms of life and can be divided into four classes by phylogenic analysis. According to available structures, Usps exist as homodimers, and genetic studies show that their cellular assignments are extensive, including functions relating to stress resistance, carbon metabolism, cellular adhesion, motility, and bacterial virulence. We approached the question of how Usps can achieve such a variety of functions in a cell by using a new procedure for statistical analysis of multiple sequence alignments, based on physicochemically related values for each amino acid residue of Usp dimer interfaces. The results predicted that Usp proteins within a class may, in addition to forming homodimers, be able to form heterodimers. Using Escherichia coli Usps as model proteins, we confirmed the existence of such interactions. We especially focused on class I UspA and UspC and demonstrated that they are able to form homo- and heterodimers in vitro and in vivo. We suggest that this ability to form both homo- and heterodimers may allow for an expansion of the functional repertoire of Usps and explains why organisms usually contain multiple usp paralogues.
7.	Larsson, Daniel, 1981- (författare) Exploring the Molecular Dynamics of Proteins and Viruses 2012 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Knowledge about structure and dynamics of the important biological macromolecules — proteins, nucleic acids, lipids and sugars — helps to understand their function. Atomic-resolution structures of macromolecules are routinely captured with X-ray crystallography and other techniques. In this thesis, simulations are used to explore the dynamics of the molecules beyond the static structures.Viruses are machines constructed from macromolecules. Crystal structures of them reveal little to no information about their genomes. In simulations of empty capsids, we observed a correlation between the spatial distribution of chloride ions in the solution and the position of RNA in crystals of satellite tobacco necrosis virus (STNV) and satellite tobacco mosaic virus (STMV). In this manner, structural features of the non-symmetric RNA could also be inferred.The capsid of STNV binds calcium ions on the icosahedral symmetry axes. The release of these ions controls the activation of the virus particle upon infection. Our simulations reproduced the swelling of the capsid upon removal of the ions and we quantified the water permeability of the capsid. The structure and dynamics of the expanded capsid suggest that the disassembly is initiated at the 3-fold symmetry axis.Several experimental methods require biomolecular samples to be injected into vacuum, such as mass-spectrometry and diffractive imaging of single particles. It is therefore important to understand how proteins and molecule-complexes respond to being aerosolized. In simulations we mimicked the dehydration process upon going from solution into the gas phase. We find that two important factors for structural stability of proteins are the temperature and the level of residual hydration. The simulations support experimental claims that membrane proteins can be protected by a lipid micelle and that a non-membrane protein could be stabilized in a reverse micelle in the gas phase. A water-layer around virus particles would impede the signal in diffractive experiments, but our calculations estimate that it should be possible to determine the orientation of the particle in individual images, which is a prerequisite for three-dimensional reconstruction.
8.	Sahin, Cagla, et al. (författare) Structural Basis for Dityrosine-Mediated Inhibition of Î±-Synuclein Fibrillization 2022 Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:27, s. 11949-11954 Tidskriftsartikel (refereegranskat)abstract Î±-Synuclein (Î±-Syn) is an intrinsically disordered protein which self-assembles into highly organized Î²-sheet structures that accumulate in plaques in brains of Parkinsonâ€™s disease patients. Oxidative stress influences Î±-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric Î±-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the Î±-Syn monomer by a factor of âˆš2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
9.	Sridhara, Sagar, 1989 (författare) Multiple structural flavors of RNase P in precursor tRNA processing. 2024 Ingår i: Wiley interdisciplinary reviews. RNA. - 1757-7012. ; 15:2 Tidskriftsartikel (refereegranskat)abstract The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
10.	Wu, Fei, 1993- (författare) Exploring Membrane Proteins within the Inner Mitochondrial and Endoplasmic Reticulum Membranes : Mitochondrial respiratory complexes and ER-localized Shr3 2024 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Membrane proteins play important roles in various life processes, for example, those in the inner mitochondrial membrane (IMM), endoplasmic reticulum (ER) membrane, and plasma membrane (PM). Oxidative phosphorylation complexes, densely packed in the IMM are crucial for energy transduction in eukaryotes. We determined three entire II2III2 IV2 supercomplex (SC) structures with 114 lipids at 2.1-2.4 Å resolution in Perkinsus marinus (P. marinus). The structures show a complete electron transfer pathway from complex II (CII) to complex IV (CIV). These architectures also reveal rotation states of the iron sulfur protein (ISP) in complex III (CIII), from one of which we observed two novel proteins that might impair the electron transfer. We also studied how the salt concentration and detergent affect the electron transfer. We determined the SC III2 IV-cytochrome c (cyt. c) cryo-EM structure at 20 mM salt concentration condition. Together with kinetic study, these data implicate that multiple cyt. c are involved in electron transfer between CIII and CIV. Our kinetic studies of CIV also indicate a native ligand bound near its K proton pathway which could be removed by detergent, leading to an increase in electron transfer rate and the activity of the enzyme. Most biogenesis of integral membrane proteins in eukaryotes is done in ER, such as the amino acid permeases (AAP), which function as amino acid transporters in the PM. Its synthesis and functional folding in Saccharomyces cerevisiae (S. cerevisiae) requires an ER membrane-localized chaperone, Shr3. We utilized a yeast growth-based genetic assay, in conjunction with a split-ubiquitin yeast two-hybrid assay, to demonstrate the selective interaction between Shr3 and nested C-terminal AAP truncations. This interaction exhibited a distinct pattern, wherein it gradually intensified and then weakened as more transmembrane helices folded. The work presented in this thesis contributions to an increased understanding of the organization and function of SCs, the effects of protein subunits, salt concentrations, and detergents on electron transfer, as well as the mechanism of Shr3 on AAP folding in the ER membrane. Together, these works have shed light on the understanding of the structure and function of several membrane proteins.
11.	Panagaki, Dimitra, et al. (författare) Nuclear envelope budding is a response to cellular stress. 2021 Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 118:30 Tidskriftsartikel (refereegranskat)abstract Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.
12.	Hemsworth, Glyn R., et al. (författare) Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut 2016 Ingår i: Open Biology. - : Royal Society of London. - 2046-2441. ; 6:7 Tidskriftsartikel (refereegranskat)abstract The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta) genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an alpha-xylosidase, a beta-glucosidase, and two alpha-L-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.
13.	Larsbrink, Johan, 1982, et al. (författare) A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin 2016 Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9:260 Tidskriftsartikel (refereegranskat)abstract Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature’s most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown.ResultsHere we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor–regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm.ConclusionsBy combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.
14.	Wu, Min, 1986, et al. (författare) Proline 411 biases the conformation of the intrinsically disordered plant UVR8 photoreceptor C27 domain altering the functional properties of the peptide 2019 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9 Tidskriftsartikel (refereegranskat)abstract UVR8 (UV RESISTANCE LOCUS 8) is a UV-B photoreceptor responsible for initiating UV-B signalling in plants. UVR8 is a homodimer in its signalling inactive form. Upon absorption of UV radiation, the protein monomerizes into its photoactivated state. In the monomeric form, UVR8 binds the E3 ubiquitin ligase COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1), triggering subsequent UV-B-dependent photomorphogenic development in plants. Recent in vivo experiments have shown that the UVR8 C-terminal region (aa 397-423; UVR8(C27)) alone is sufficient to regulate the activity of COP1. In this work, CD spectroscopy and NMR experiments showed that the UVR8(C27) domain was non-structured but gained secondary structure at higher temperatures leading to increased order. Bias-exchange metadynamics simulations were also performed to evaluate the free energy landscape of UVR8(C27). An inverted free energy landscape was revealed, with a disordered structure in the global energy minimum. Flanking the global energy minimum, more structured states were found at higher energies. Furthermore, stabilization of the low energy disordered state was attributed to a proline residue, P411, as evident from P411A mutant data. P411 is also a key residue in UVR8 binding to COP1. UVR8(C27) is therefore structurally competent to function as a molecular switch for interaction of UVR8 with different binding partners since at higher free energies different structural conformations are being induced in this peptide. P411 has a key role for this function.
15.	Zeng, Jiao, et al. (författare) High-resolution structure of a fish aquaporin reveals a novel extracellular fold 2022 Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12 Tidskriftsartikel (refereegranskat)abstract Aquaporins are protein channels embedded in the lipid bilayer in cells from all organisms on earth that are crucial for water homeostasis. In fish, aquaporins are believed to be important for osmoregulation; however, the molecular mechanism behind this is poorly understood. Here, we present the first structural and functional characterization of a fish aquaporin; cpAQP1aa from the fresh water fish climbing perch (Anabas testudineus), a species that is of high osmoregulatory interest because of its ability to spend time in seawater and on land. These studies show that cpAQP1aa is a water-specific aquaporin with a unique fold on the extracellular side that results in a constriction region. Functional analysis combined with molecular dynamic simulations suggests that phosphorylation at two sites causes structural perturbations in this region that may have implications for channel gating from the extracellular side.
16.	Purayil, Siju, et al. (författare) Neuropeptides in the Antennal Lobe of the Yellow Fever Mosquito, Aedes aegypti. 2014 Ingår i: Journal of Comparative Neurology. - : Wiley. - 0021-9967 .- 1096-9861. ; 522, s. 592-608 Tidskriftsartikel (refereegranskat)abstract For many insects, including mosquitoes, olfaction is the dominant modality regulating their behavioral repertoire. Many neurochemicals modulate olfactory information in the central nervous system, including the primary olfactory center of insects, the antennal lobe. The most diverse and versatile neurochemicals in the insect nervous system are found in the neuropeptides. In the present study, we analyzed neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti, a major vector of arboviral diseases. Direct tissue profiling of the antennal lobe by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of 28 mature products from 10 different neuropeptide genes. In addition, immunocytochemical techniques were used to describe the cellular location of the products of up to seven of these genes within the antennal lobe. Allatostatin A, allatotropin, SIFamide, FMRFamide-related peptides, short neuropeptide F, myoinhibitory peptide, and tachykinin-related peptides were found to be expressed in local interneurons and extrinsic neurons of the antennal lobe. Building on these results, we discuss the possible role of neuropeptide signaling in the antennal lobe of Ae. aegypti. J. Comp. Neurol. 522:592-608, 2014. (c) 2013 Wiley Periodicals, Inc.
17.	D'Angiolo, M., et al. (författare) A yeast living ancestor reveals the origin of genomic introgressions 2020 Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 587, s. 420-425 Tidskriftsartikel (refereegranskat)abstract A yeast clonal descendant of an ancient hybridization event is identified and sheds light on the early evolution of the Saccharomyces cerevisiae Alpechin lineage and its abundant Saccharomyces paradoxus introgressions. Genome introgressions drive evolution across the animal(1), plant(2) and fungal(3) kingdoms. Introgressions initiate from archaic admixtures followed by repeated backcrossing to one parental species. However, how introgressions arise in reproductively isolated species, such as yeast(4), has remained unclear. Here we identify a clonal descendant of the ancestral yeast hybrid that founded the extant Saccharomyces cerevisiae Alpechin lineage(5), which carries abundant Saccharomyces paradoxus introgressions. We show that this clonal descendant, hereafter defined as a 'living ancestor', retained the ancestral genome structure of the first-generation hybrid with contiguous S. cerevisiae and S. paradoxus subgenomes. The ancestral first-generation hybrid underwent catastrophic genomic instability through more than a hundred mitotic recombination events, mainly manifesting as homozygous genome blocks generated by loss of heterozygosity. These homozygous sequence blocks rescue hybrid fertility by restoring meiotic recombination and are the direct origins of the introgressions present in the Alpechin lineage. We suggest a plausible route for introgression evolution through the reconstruction of extinct stages and propose that genome instability allows hybrids to overcome reproductive isolation and enables introgressions to emerge.
18.	Stenlid, Jan (författare) The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes 2012 Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 336, s. 1715-1719 Tidskriftsartikel (refereegranskat)abstract Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non-lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.
19.	Xu, Bo, 1980- (författare) Evolutionary and Pharmacological Studies of NPY and QRFP Receptors 2014 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract The neuropeptide Y (NPY) system consists of 3-4 peptides and 4-7 receptors in vertebrates. It has powerful effects on appetite regulation and is involved in many other biological processes including blood pressure regulation, bone formation and anxiety. This thesis describes studies of the evolution of the NPY system by comparison of several vertebrate species and structural studies of the human Y2 receptor, which reduces appetite, to identify amino acid residues involved in peptide-receptor interactions.The NPY system was studied in zebrafish (Danio rerio), western clawed frog (Xenopus tropicalis), and sea lamprey (Petromyzon marinus). The receptors were cloned and functionally expressed and their pharmacological profiles were determined using the native peptides in either binding studies or a signal transduction assay. Some peptide-receptor preferences were observed, indicating functional specialization.A receptor family closely related to the NPY receptors, called the QRFP receptors, was investigated. A QRFP receptor was cloned from amphioxus, Branchistoma floridae, showing that the receptor arose before the origin of the vertebrates. Evolutionary studies demonstrated that the ancestral vertebrate had as many as four QRFP receptors, only one of which remains in mammals today. This correlates with the NPY receptor family, located in the same chromosomal regions, which had seven members in the ancestral vertebrate but only 4-5 in living mammals. Some vertebrates have considerably more complex NPY and QRFP receptor systems than humans and other mammals.Two studies investigated interactions of NPY-family peptides with the human Y2 receptor. Candidate residues, selected based on structural modeling and docking, were mutated to disrupt possible interactions with peptide ligands. The modified receptors were expressed in cultured cells and investigated by measuring binding and functional responses. Several receptor residues were found to influence peptide-receptor interactions, some of which are involved in maintaining receptor structure. In a pilot study, the kinetics of peptide-receptor interaction were found to be very slow, of the order several hours.In conclusion, this thesis clarifies evolutionary relationships for the complex NPY and QRFP peptide-receptor systems and improves the structural models of the human NPY-family receptors, especially Y2. These results will hopefully facilitate drug design for targeting of NPY-family receptors.
20.	Allison, Timothy M., et al. (författare) Complementing machine learning‐based structure predictions with native mass spectrometry 2022 Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:6 Tidskriftsartikel (refereegranskat)abstract The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.
21.	Conti, Luca, et al. (författare) Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation 2016 Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6 Tidskriftsartikel (refereegranskat)abstract Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.
22.	Fröhlich, Christopher, et al. (författare) Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1. 2020 Ingår i: The Journal of antimicrobial chemotherapy. - : Oxford University Press (OUP). - 1460-2091 .- 0305-7453. ; 75:9, s. 2554-2563 Tidskriftsartikel (refereegranskat)abstract MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure-activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure.To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1.The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1).Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity.These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.
23.	Khaji, Erfan, et al. (författare) 3D protein structure prediction using Imperialist Competitive algorithm and half sphere exposure prediction 2016 Ingår i: Journal of Theoretical Biology. - : Elsevier BV. - 0022-5193. ; 391, s. 81-87 Tidskriftsartikel (refereegranskat)abstract Predicting the native structure of proteins based on half-sphere exposure and contact numbers has been studied deeply within recent years. Online predictors of these vectors and secondary structures of amino acids sequences have made it possible to design a function for the folding process. By choosing variant structures and directs for each secondary structure, a random conformation can be generated, and a potential function can then be assigned. Minimizing the potential function utilizing meta-heuristic algorithms is the final step of finding the native structure of a given amino acid sequence. In this work, Imperialist Competitive algorithm was used in order to accelerate the process of minimization. Moreover, we applied an adaptive procedure to apply revolutionary changes. Finally, we considered a more accurate tool for prediction of secondary structure. The results of the computational experiments on standard benchmark show the superiority of the new algorithm over the previous methods with similar potential function. © 2015 Elsevier Ltd.
24.	Landreh, Michael, et al. (författare) Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters 2017 Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8 Tidskriftsartikel (refereegranskat)abstract Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.
25.	Papadopoulos, Evangelos, et al. (författare) Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli 2012 Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1824:12, s. 1401-1408 Tidskriftsartikel (refereegranskat)abstract The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K-a >= 10(17) M-1 at 23 degrees C, pH 7.0).
26.	Reddy, Hemanth K.N., et al. (författare) Electron cryo-microscopy of bacteriophage PR772 reveals the elusive vertex complex and the capsid architecture 2019 Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8 Tidskriftsartikel (refereegranskat)abstract Bacteriophage PR772, a member of the Tectiviridae family, has a 70 nm diameter icosahedral protein capsid that encapsulates a lipid membrane, dsDNA, and various internal proteins. An icosahedrally averaged CryoEM reconstruction of the wild-type virion and a localized reconstruction of the vertex region reveal the composition and the structure of the vertex complex along with new protein conformations that play a vital role in maintaining the capsid architecture of the virion. The overall resolution of the virion is 2.75 angstrom, while the resolution of the protein capsid is 2.3 angstrom. The conventional penta-symmetron formed by the capsomeres is replaced by a large vertex complex in the pseudo T = 25 capsid. All the vertices contain the host-recognition protein, P5; two of these vertices show the presence of the receptor-binding protein, P2. The 3D structure of the vertex complex shows interactions with the viral membrane, indicating a possible mechanism for viral infection.
27.	Rozman Grinberg, Inna, et al. (författare) Class Id ribonucleotide reductase utilizes a Mn-2(IV,III) cofactor and undergoes large conformational changes on metal loading 2019 Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:6, s. 863-877 Tidskriftsartikel (refereegranskat)abstract Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn-2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn-2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn-2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O2 center dot-). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.
28.	Höög, Johanna L, 1979, et al. (författare) Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy : Diversity of extracellular vesicles in human ejaculates 2015 Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 4 Tidskriftsartikel (refereegranskat)abstract Human ejaculates contain extracellular vesicles (EVs), that to a large extent are considered to originate from the prostate gland, and are often denominated ‘‘prostasomes.’’ These EVs are important for human fertility, for example by promoting sperm motility and by inducing immune tolerance of the female immune system to the spermatozoa. So far, the EVs present in human ejaculate have not been studied in their native state, inside the seminal fluid without prior purification and isolation procedures. Using cryo-electron microscopy and tomography, we performed a comprehensive inventory of human ejaculate EVs. The sample was neither centrifuged, fixed, filtered or sectioned, nor were heavy metals added. Approximately 1,500 extracellular structures were imaged and categorized. The extracellular environment of human ejaculate was found to be diverse, with 5 major subcategories of EVs and 6 subcategories of extracellular membrane compartments, including lamellar bodies. Furthermore, 3 morphological features, including electron density, double membrane bilayers and coated surface, are described in all subcategories. This study reveals that the extracellular environment in human ejaculate is multifaceted. Several novel morphological EV subcategories are identified and clues to their cellular origin may be found in their morphology. This inventory is therefore important for developing future experimental approaches, and to interpret previously published data to understand the role of EVs for human male fertility.
29.	Kang, Wenjing, 1988- (författare) microRNAs: from biogenesis to organismal tracing 2020 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract MicroRNAs (miRNAs) are short noncoding RNAs of around 22 nucleotides in length, which help to shape the expression of most mRNAs. Perturbation of miRNA expression has revealed a variety of defects in development, cell specification, physiology and behavior. This thesis focuses on two topics of miRNA: identification of structural features that influence miRNA biogenesis (Paper I) and application of taxonomical marker miRNAs to resolve organismal origin of samples (Paper II and III).The current model of miRNA hairpin biogenesis has limited information content and appears to be incomplete. In paper I, we apply a novel high-throughput screening method to profile the optimal structure of miRNA hairpins for efficient and precise miRNA biogenesis. The optimal structure consists of tight and loose local structures across the hairpin, which reflects the constraints of biogenesis proteins. We find that miRNA hairpins with stable lower basal stem are more efficiently processed and have a higher expression level in tissues of 20 animal species. We address that the structural features - which have been largely neglected in the current model - are in fact as important as the well-known sequence motifs.New miRNAs are continuously added over evolutionary time and are rarely secondarily lost, making them ideal taxonomical markers. In paper II, we demonstrate as a proof-of-principle that miRNAs can be used to trace biological sample back to the lineage or even species of origin. Based on the marker miRNAs, we develop miRTrace, the first software to accurately trace miRNA sequences back to their taxonomical origin. The method can sensitively identify the origin of single cells and detect parasitic nematode RNA in mammalian host blood sample. In paper III, we apply miRNA tracing to address a controversial question about the origin of the exogenous plant miRNAs (xenomiRs) found in human samples, and which have been proposed to regulate human gene expression. Our computational and experimental results provide evidence that xenomiRs are derived from technical artifacts rather than dietary intake.
30.	Zabeo, Davide, 1992, et al. (författare) A lumenal interrupted helix in human sperm tail microtubules 2018 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8 Tidskriftsartikel (refereegranskat)abstract Eukaryotic flagella are complex cellular extensions involved in many human diseases gathered under the term ciliopathies. Currently, detailed insights on flagellar structure come mostly from studies on protists. Here, cryo-electron tomography (cryo-ET) was performed on intact human spermatozoon tails and showed a variable number of microtubules in the singlet region (inside the end-piece). Inside the microtubule plus end, a novel left-handed interrupted helix which extends several micrometers was discovered. This structure was named Tail Axoneme Intra-Lumenal Spiral (TAILS) and binds directly to 11 protofilaments on the internal microtubule wall, in a coaxial fashion with the surrounding microtubule lattice. It leaves a gap over the microtubule seam, which was directly visualized in both singlet and doublet microtubules. We speculate that TAILS may stabilize microtubules, enable rapid swimming or play a role in controlling the swimming direction of spermatozoa.
31.	Borisova, Anna, et al. (författare) Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity 2015 Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290, s. 22955-22969 Tidskriftsartikel (refereegranskat)abstract The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose beta-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+-center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9Cenabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4- oxidized products show an intermediate situation.
32.	Isberg, Elin, et al. (författare) Comparative Study of Antennal and Maxillary Palp Olfactory Sensilla of Female Biting Midges (Diptera: Ceratopogonidae: Culicoides) in the Context of Host Preference and Phylogeny 2013 Ingår i: Journal of Medical Entomology. - 0022-2585. ; 50, s. 485-492 Tidskriftsartikel (refereegranskat)abstract Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of disease, including bluetongue and African horse sickness. Host preference of these insects is primarily regulated by olfactory cues, detected by olfactory sensilla on the antennae and maxillary palps. In this study, we analyzed the sensillum repertoire of biting midge species with known host preferences. Five different morphological sensillum types, sensilla trichodea, s. chaetica, s. ampullacea, s. coeloconica, and grooved peg sensilla, were present on the antennae of all species. In addition sensilla basiconica were present on the maxillary palps. We found that the numbers of short blunt-tipped s. trichodea, s. coeloconica, and s. basiconica are significantly higher in the ornithophilic Culicoides festivipennis (Kieffer) compared with the mammalophilic Culicoides obsoletus (Meigen) and Culicoides chiopterus (Meigen). In contrast, we found that the mammalophilic Culicoides pulicaris (L.) and the opportunistic Culicoides punctatus (Meigen) have intermediate numbers of these sensillum types. Comparison with available data from other species strongly suggests that these differences in the number of specific sensillum types, in general, are a reflection of host preference and not of phylogeny. We discuss the putative function of the individual sensillum types in relation to host volatile detection.
33.	Kvarnheden, Anders (författare) The Peptide AmPep1 Derived from Amaranth Recognizes the Replication Hairpin of TYLCV Disturbing Its Replication Process in Host Plants 2019 Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 67, s. 9241-9253 Tidskriftsartikel (refereegranskat)abstract Antiviral compounds targeting viral replicative processes have been studied as an alternative for the control of begomoviruses. Previously, we have reported that the peptide AmPep1 has strong affinity binding to the replication origin sequence of tomato yellow leaf curl virus (TYLCV). In this study, we describe the mechanism of action of this peptide as a novel alternative for control of plant-infecting DNA viruses. When AmPep1 was applied exogenously to tomato and Nicotiana benthamiana plants infected with TYLCV, a decrease in the synthesis of the two viral DNA strands (CS and VS) was observed, with a consequent delay in the development of disease progress in treated plants. The chemical mechanism of action of AmPep1 was deduced using Raman spectroscopy and molecular modeling showing the formation of chemical interactions such as H bonds and electrostatic interactions and the formation of pi-pi interactions between both biomolecules contributing to tampering with the viral replication.
34.	Munke, Anna, et al. (författare) Primordial Capsid and Spooled ssDNA Genome Structures Unravel Ancestral Events of Eukaryotic Viruses 2022 Ingår i: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 13:4 Tidskriftsartikel (refereegranskat)abstract Marine algae viruses are important for controlling microorganism communities in the marine ecosystem and played fundamental roles during the early events of viral evolution. Here, we have focused on one major group of marine algae viruses, the single-stranded DNA (ssDNA) viruses from the Bacilladnaviridae family. We present the capsid structure of the bacilladnavirus Chaetoceros tenuissimus DNA virus type II (CtenDNAV-II), determined at 2.4-Å resolution. A structure-based phylogenetic analysis supported the previous theory that bacilladnaviruses have acquired their capsid protein via horizontal gene transfer from a ssRNA virus. The capsid protein contains the widespread virus jelly-roll fold but has additional unique features; a third β-sheet and a long C-terminal tail. Furthermore, a low-resolution reconstruction of the CtenDNAV-II genome revealed a partially spooled structure, an arrangement previously only described for dsRNA and dsDNA viruses. Together, these results exemplify the importance of genetic recombination for the emergence and evolution of ssDNA viruses and provide important insights into the underlying mechanisms that dictate genome organization.IMPORTANCE Single-stranded DNA (ssDNA) viruses are an extremely widespread group of viruses that infect diverse hosts from all three domains of life, consequently having great economic, medical, and ecological importance. In particular, bacilladnaviruses are highly abundant in marine sediments and greatly influence the dynamic appearance and disappearance of certain algae species. Despite the importance of ssDNA viruses and the last couple of years' advancements in cryo-electron microscopy, structural information on the genomes of ssDNA viruses remains limited. This paper describes two important achievements: (i) the first atomic structure of a bacilladnavirus capsid, which revealed that the capsid protein gene presumably was acquired from a ssRNA virus in early evolutionary events; and (ii) the structural organization of a ssDNA genome, which retains a spooled arrangement that previously only been observed for double-stranded viruses.
35.	Andersson, Marlene, et al. (författare) Biomimetic spinning of artificial spider silk from a chimeric minispidroin 2017 Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 13:3, s. 262- Tidskriftsartikel (refereegranskat)abstract Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.
36.	Aurelius, Oskar, et al. (författare) The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site 2015 Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:7 Tidskriftsartikel (refereegranskat)abstract Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.
37.	Costeira-Paulo, Joana, et al. (författare) Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase 2018 Ingår i: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 25:3, s. 309-317 Tidskriftsartikel (refereegranskat)abstract The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
38.	Diamanti, Riccardo, et al. (författare) Comparative structural analysis provides new insights into the function of R2-like ligand-binding oxidase 2022 Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 596:12, s. 1600-1610 Tidskriftsartikel (refereegranskat)abstract R2-like ligand-binding oxidase (R2lox) is a ferritin-like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross-link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry-based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.
39.	Grāve, Kristīne, et al. (författare) The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron 2020 Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 25:4, s. 571-582 Tidskriftsartikel (refereegranskat)abstract Correct protein metallation in the complex mixture of the cell is a prerequisite for metalloprotein function. While some metals, such as Cu, are commonly chaperoned, specificity towards metals earlier in the Irving-Williams series is achieved through other means, the determinants of which are poorly understood. The dimetal carboxylate family of proteins provides an intriguing example, as different proteins, while sharing a common fold and the same 4-carboxylate 2-histidine coordination sphere, are known to require either a Fe/Fe, Mn/Fe or Mn/Mn cofactor for function. We previously showed that the R2lox proteins from this family spontaneously assemble the heterodinuclear Mn/Fe cofactor. Here we show that the class Ib ribonucleotide reductase R2 protein from Bacillus anthracis spontaneously assembles a Mn/Mn cofactor in vitro, under both aerobic and anoxic conditions, when the metal-free protein is subjected to incubation with Mn-II and Fe-II in equal concentrations. This observation provides an example of a protein scaffold intrinsically predisposed to defy the Irving-Williams series and supports the assumption that the Mn/Mn cofactor is the biologically relevant cofactor in vivo. Substitution of a second coordination sphere residue changes the spontaneous metallation of the protein to predominantly form a heterodinuclear Mn/Fe cofactor under aerobic conditions and a Mn/Mn metal center under anoxic conditions. Together, the results describe the intrinsic metal specificity of class Ib RNR and provide insight into control mechanisms for protein metallation.
40.	Jacobson, Mark J., et al. (författare) Purification, Modeling, and Analysis of Botulinum Neurotoxin Subtype A5 (BoNT/A5) from Clostridium botulinum Strain A661222 2011 Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 77:12, s. 4217-4222 Tidskriftsartikel (refereegranskat)abstract A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences-specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.
41.	Johansson, Renzo, et al. (författare) Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones 2016 Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 24:6, s. 906-917 Tidskriftsartikel (refereegranskat)abstract Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.
42.	Konasani, Venkat Rao, 1981, et al. (författare) A novel ulvan lyase family with broad-spectrum activity from the ulvan utilisation loci of Formosa agariphila KMM 3901 2018 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8 Tidskriftsartikel (refereegranskat)abstract Ulvan, which is one of the major structural polysaccharides of the cell walls of green macroalgae, is degraded by ulvan lyases via the beta-elimination mechanism with the release of oligosaccharides that have unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronic acid (Delta) at the non-reducing end. These ulvan lyases belong to the PL24 or PL25 or PL28 family in the CAZy database. In this study, we identify and biochemically characterise a periplasmic novel broad-spectrum ulvan lyase from Formosa agariphila KMM 3901. The lyase was overexpressed in Escherichia coli, and the purified recombinant enzyme depolymerised ulvan in an endolytic manner with a K-m of 0.77 mg/ml, and displayed optimum activity at 40 degrees C and pH 8. This lyase also degraded heparan sulphate and chondroitin sulphate. Detailed analyses of the end-products of the enzymatic degradation of ulvan using H-1- and C-13-NMR and LC-MS revealed an unsaturated disaccharide (Delta Rha3S) and a tetrasaccharide (Delta Rha3S-Xyl-Rha) as the principal end-products. In contrast to the previously described ulvan lyases, this novel lyase is mostly composed of alpha-helices that form an (alpha/alpha)(6) incomplete toroid domain and displays a remarkably broad-spectrum activity. This novel lyase is the first member of a new family of ulvan lyases.
43.	Kutin, Yuri, et al. (författare) Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins 2016 Ingår i: Journal of Inorganic Biochemistry. - : Elsevier BV. - 0162-0134 .- 1873-3344. ; 162, s. 164-177 Tidskriftsartikel (refereegranskat)abstract A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of dass Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is undear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn-II and Fe-II. Using EPR and Mfissbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn-II promotes heterobimetallic cofactor assembly. In the absence of Fe-II, R2c cooperatively binds Mn-II at both metal sites, whereas R2lox does not readily bind Mn-II at either site. Heterometallic cofactor assembly is favored at substoichiometric Feu concentrations in R2lox. Fe-II and Mn-II likely bind to the protein in a stepwise fashion, with Feu binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn-II by Fe-II at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn-II/Fe-II concentrations in the host organisms from which they were isolated.
44.	Schriever, Karen (författare) Sequence- and structure guided engineering of proteins and enzymes for biotechnology and health applications 2023 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Proteins are highly diverse and sophisticated biomolecules that represent a cornerstone of biological structure and function and have been exploited in man-made applications for thousands of years. Those proteins that facilitate chemical reactions at physiologically relevant time-scales are referred to as enzymes. Understanding the connections between proteins’ functions and their structures, mechanisms and evolution allows to engineer them towards desired properties for various applications. The aim of the work presented in this thesis is to assess different protein engineering approaches and workflows in the context of health and biotechnology applications. Four proteins were studied and/or engineered towards different outcomes using either sequence‑based information, structural information or a combination thereof. In paper I a sequence-based approach was applied to optimise vaccine candidates for severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Specifically, ancestral sequence reconstruction was used to generate highly stable and soluble antigens that could be produced in high quantities in a low-throughput and structure‑independent manner. These ancestral antigens interacted with antibodies from recovered patients and served as scaffolds to host a domain of the extant antigen to further enhance antibody engagement. Paper II and III applied enzyme engineering to terpene cyclases in a health and biocatalysis context, respectively. In paper II a structure-based approach was used to understand the fundamental principles underlying the catalytic mechanism of an enzyme in human steroid metabolism. Specifically, solvent access tunnels were identified and modified to probe the role of activation entropy in human oxidosqualene cyclase, which drastically modified the temperature dependence of catalysis. This finding may also have implications for engineering related plant enzymes for production of industrially relevant compounds in heterologous hosts. In paper III sequence- and structure based approaches were used together to engineer substrate specificity in a promiscuous bacterial terpene cyclase. Specifically, the structure of a stable reconstructed ancestor of spiroviolene synthase was determined in order to understand the molecular basis of substrate promiscuity and engineer highly selective variants that retained thermostability. The presented workflow is relevant for engineering these enzymes as biocatalysts for production of terpene-based high value compounds. In paper IV the metabolite regulation of a flux-controlling enzyme in the Calvin cycle was studied to eventually engineer it for enhanced growth of autotrophic production hosts. Specifically, interactions between a bifunctional cyanobacterial fructose‑1,6-bisphosphatase and a panel of metabolites were identified using a proteomics approach and verified by in vitro experiments. A synergistic regulation involving the enzyme’s redox state and glyceraldehyde 3‑phosphate was discovered, which has implications for integrated metabolic and enzyme engineering approaches involving this biocatalyst. In summary, the results presented herein highlight the utility of integrating several different engineering approaches for proteins used in health and biotechnology applications.
45.	Seely, Savannah M., et al. (författare) Molecular basis of the pleiotropic effects by the antibiotic amikacin on the ribosome 2023 Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1 Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3’-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties.
46.	Zhuravleva, Anastasia, 1979, et al. (författare) Divided evolution: a scheme for suppression of line broadening induced by conformational exchange 2008 Ingår i: Journal of the American Chemical Society. - 0002-7863. ; 130:11, s. 3260-3260 Tidskriftsartikel (refereegranskat)abstract We present a new signal acquisition scheme termed D-evolution that improves NMR spectra for systems undergoing conformational exchange in the intermediate time scale. The new approach separates time scales of the conformational exchange and chemical shift evolution. This reduces line broadening by avoiding the situation of intermediate exchange. The chemical shift evolution is divided into a number of short intervals interfaced by Carr-Purcell-Meiboom-Gill (CPMG) blocks. We demonstrate this method for several test systems such as small molecules, cyclohexane, and acetonitrile as well as two globular proteins, azurin and protein-L.
47.	Ezzedine, Jade A., et al. (författare) Adaptive traits of cysts of the snow alga Sanguina nivaloides unveiled by 3D subcellular imaging 2023 Ingår i: Nature Communications. - 2041-1723. ; 14:1 Tidskriftsartikel (refereegranskat)abstract Sanguina nivaloides is the main alga forming red snowfields in high mountains and Polar Regions. It is non-cultivable. Analysis of environmental samples by X-ray tomography, focused-ion-beam scanning-electron-microscopy, physicochemical and physiological characterization reveal adaptive traits accounting for algal capacity to reside in snow. Cysts populate liquid water at the periphery of ice, are photosynthetically active, can survive for months, and are sensitive to freezing. They harbor a wrinkled plasma membrane expanding the interface with environment. Ionomic analysis supports a cell efflux of K+, and assimilation of phosphorus. Glycerolipidomic analysis confirms a phosphate limitation. The chloroplast contains thylakoids oriented in all directions, fixes carbon in a central pyrenoid and produces starch in peripheral protuberances. Analysis of cells kept in the dark shows that starch is a short-term carbon storage. The biogenesis of cytosolic droplets shows that they are loaded with triacylglycerol and carotenoids for long-term carbon storage and protection against oxidative stress.
48.	Fitzer, Susan, et al. (författare) Established and emerging techniques for characterising the formation, structure and performance of calcified structures under ocean acidification 2019 Ingår i: Oceanography and Marine Biology: An Annual Review 57. - Boca Raton, FL, USA : CRC Press. - 0078-3218. - 9780367134150 ; , s. 89-126 Bokkapitel (refereegranskat)abstract Ocean acidification (OA) is the decline in seawater pH and saturation levels of calcium carbonate (CaCO3) minerals that has led to concerns for calcifying organisms such as corals, oysters and mussels because of the adverse effects of OA on their biomineralisation, shells and skeletons. A range of cellular biology, geochemistry and materials science approaches have been used to explore biomineralisation. These techniques have revealed that responses to seawater acidification can be highly variable among species, yet the underlying mechanisms remain largely unresolved. To assess the impacts of global OA, researchers will need to apply a range of tools developed across disciplines, many of which are emerging and have not yet been used in this context. This review outlines techniques that could be applied to study OA-induced alterations in the mechanisms of biomineralisation and their ultimate effects on shells and skeletons. We illustrate how to characterise, quantify and monitor the process of biomineralisation in the context of global climate change and OA. We highlight the basic principles, as well as the advantages and disadvantages, of established, emerging and future techniques for OA researchers. A combination of these techniques will enable a holistic approach and better understanding of the potential impact of OA on biomineralisation and its consequences for marine calcifiers and associated ecosystems.
49.	Hallberg, Eric, et al. (författare) Sensilla and proprioceptors 2003 Ingår i: Lepidoptera, Moths and Butterflies : Volume 2: Morphology, Physiology, and Development - Volume 2: Morphology, Physiology, and Development. - : DE GRUYTER. - 9783110893724 - 3110162105 - 9783110162103 ; , s. 267-288 Bokkapitel (refereegranskat)
50.	Höög, Johanna L, 1979, et al. (författare) 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction : Electron Tomography of the Flagella Connector 2016 Ingår i: PLoS Neglected Tropical Diseases. - : Public Library of Science (PLoS). - 1935-2727 .- 1935-2735. ; 10:1 Tidskriftsartikel (refereegranskat)abstract Background Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC) is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility. Methodology/Principal Findings We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum. Conclusions/Significance The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

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