| 1. |
- Andersson, Marlene, et al.
(författare)
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Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains
- 2014
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 12:8, s. e1001921-
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Tidskriftsartikel (refereegranskat)abstract
- Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive beta-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO(2)) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.
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| 2. |
- Arndt, Tina, et al.
(författare)
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Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.
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| 3. |
- Gault, Joseph, et al.
(författare)
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Mass Spectrometry Reveals the Direct Action of a Chemical Chaperone
- 2018
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Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 9:14, s. 4082-4086
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Tidskriftsartikel (refereegranskat)abstract
- Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine-N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine-N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.
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| 4. |
- Kaldmae, Margit, et al.
(författare)
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Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction
- 2019
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 30:8, s. 1385-1388
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Tidskriftsartikel (refereegranskat)abstract
- Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation.
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| 5. |
- Kaldmäe, Margit, et al.
(författare)
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A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder
- 2022
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 30:5, s. 733-742, e1-e7
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Tidskriftsartikel (refereegranskat)abstract
- Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.
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| 6. |
- Kronqvist, Nina, et al.
(författare)
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A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
- 2008
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 21:4, s. 247-255
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Tidskriftsartikel (refereegranskat)abstract
- Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.
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| 7. |
- Kronqvist, Nina, et al.
(författare)
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Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
- 2011
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 24:4, s. 385-396
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Tidskriftsartikel (refereegranskat)abstract
- Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.
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| 8. |
- Kronqvist, Nina, et al.
(författare)
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Efficient protein production inspired by how spiders make silk
- 2017
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT star) that is pH insensitive, stabilized and hypersoluble compared to wildtype NT. NT star-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT star enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT star also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
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| 9. |
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| 10. |
- Kronqvist, Nina, et al.
(författare)
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Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation
- 2014
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 5:1, s. 3254-
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Tidskriftsartikel (refereegranskat)abstract
- The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.
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