| 1. |
- Young, Gavin, et al.
(författare)
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Quantitative mass imaging of single biological macromolecules
- 2018
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Ingår i: Science. - : American Association for the Advancement of Science. - 0036-8075 .- 1095-9203. ; 360:6387, s. 423-427
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Tidskriftsartikel (refereegranskat)abstract
- Careful measurements of light scattering can provide information on individual macromolecules and complexes. Young et al. used a light-scattering approach for accurate mass determination of proteins as small as 20 kDa (see the Perspective by Lee and Klenerman). Movies of protein complex association and dissociation were analyzed to extract biophysical parameters from single molecules and assemblies without labeling. Using this approach, the authors determined in vitro kinetics of fibril and aggregate growth and association constants for a complex protein-glycoprotein assembly.Science, this issue p. 423; see also p. 378The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.
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| 2. |
- Alex, Amal, et al.
(författare)
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Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
- 2019
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.
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| 3. |
- Danielsson, Jens, et al.
(författare)
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Thermodynamics of protein destabilization in live cells
- 2015
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:40, s. 12402-12407
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Tidskriftsartikel (refereegranskat)abstract
- Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a beta-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 degrees C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized interaction landscape of the cellular interior.
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