| 11. |
- Conti, Luca, et al.
(author)
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Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation
- 2016
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In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Journal article (peer-reviewed)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.
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| 12. |
- Höög, Johanna L, 1979, et al.
(author)
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Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy : Diversity of extracellular vesicles in human ejaculates
- 2015
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In: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 4
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Journal article (peer-reviewed)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.
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| 13. |
- Khaji, Erfan, et al.
(author)
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3D protein structure prediction using Imperialist Competitive algorithm and half sphere exposure prediction
- 2016
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In: Journal of Theoretical Biology. - : Elsevier BV. - 0022-5193. ; 391, s. 81-87
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Journal article (peer-reviewed)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.
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| 14. |
- Reddy, Hemanth K.N., et al.
(author)
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Electron cryo-microscopy of bacteriophage PR772 reveals the elusive vertex complex and the capsid architecture
- 2019
-
In: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Journal article (peer-reviewed)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.
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| 15. |
- Rozman Grinberg, Inna, et al.
(author)
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Class Id ribonucleotide reductase utilizes a Mn-2(IV,III) cofactor and undergoes large conformational changes on metal loading
- 2019
-
In: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:6, s. 863-877
-
Journal article (peer-reviewed)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.
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| 16. |
- Zabeo, Davide, 1992, et al.
(author)
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A lumenal interrupted helix in human sperm tail microtubules
- 2018
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
-
Journal article (peer-reviewed)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.
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| 17. |
- Newton, Matilda S., et al.
(author)
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Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes
- 2017
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:8, s. 4727-4732
-
Journal article (peer-reviewed)abstract
- New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (βα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (βα)8 barrel as an inherently evolvable protein scaffold.
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| 18. |
- Andersson, Marlene, et al.
(author)
-
Biomimetic spinning of artificial spider silk from a chimeric minispidroin
- 2017
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In: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 13:3, s. 262-
-
Journal article (peer-reviewed)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.
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| 19. |
- Borisova, Anna, et al.
(author)
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Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity
- 2015
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290, s. 22955-22969
-
Journal article (peer-reviewed)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.
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| 20. |
- Fitzer, Susan, et al.
(author)
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Established and emerging techniques for characterising the formation, structure and performance of calcified structures under ocean acidification
- 2019
-
In: Oceanography and Marine Biology: An Annual Review 57. - Boca Raton, FL, USA : CRC Press. - 0078-3218. - 9780367134150 ; , s. 89-126
-
Book chapter (peer-reviewed)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.
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