| 1. |
- Manz, Christoph, et al.
(författare)
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Energy Landscape Analysis of the Full-Length SAM-I Riboswitch using Single-Molecule FRET Spectroscopy
- 2018
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495.
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Konferensbidrag (refereegranskat)abstract
- Binding of the ligand S-adenosyl-L-methionine (SAM) produces major structural changes in the SAM-I riboswitch (RS) and thereby regulates gene expression via transcription termination. As yet, the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I RS have not been deeply investigated. We have studied its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy. smFRET histograms of differently FRET-labeled constructs were so complicated that they could only be resolved with the help of kinetic experiments on immobilized riboswitches and hidden Markov modeling (HMM) analysis. At least four conformational states were identified, both in the presence and the absence of SAM. We determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds were found to coexist under all conditions; SAM binding induced only a gradual increase in the population of terminator states. Conformational transitions were much faster with bound SAM, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.
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| 2. |
- Klapper, Yvonne, et al.
(författare)
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Low affinity binding of plasma proteins to lipid-coated quantum dots as observed by in situ fluorescence correlation spectroscopy
- 2015
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Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3364 .- 2040-3372. ; 7:22, s. 9980-9984
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Tidskriftsartikel (refereegranskat)abstract
- Protein binding to lipid-coated nanoparticles has been pursued quantitatively by using fluorescence correlation spectroscopy. The binding of three important plasma proteins to lipid-enwrapped quantum dots (QDs) shows very low affinity, with an apparent dissociation coefficient in the range of several hundred micromolar. Thus, the tendency to adsorb is orders of magnitude weaker than for QDs coated with dihydrolipoic acid.
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| 3. |
- Klapper, Yvonne, et al.
(författare)
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Mediation of a non-proteolytic activation of complement component C3 by phospholipid vesicles
- 2014
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Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 35:11, s. 3688-3696
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Tidskriftsartikel (refereegranskat)abstract
- Liposomes are becoming increasingly important as drug delivery systems, to target a drug to specific cells and tissues and thereby protecting the recipient from toxic effects of the contained drug. Liposome preparations have been described to activate complement. In this study, we have investigated complement activation triggered by neutral dimyristoyl-phosphocholine (DMPC) liposomes in human plasma and whole-blood systems. Incubation in plasma led to the generation of complement activation products (C3a and sC5b-9). Unexpectedly, investigations of surface-bound C3 revealed contact activated, conformationally changed C3 molecules on the liposomes. These changes were characterized by Western blotting with C3 monoclonal antibodies, and by incubating liposomes with purified native C3 and factors I and H. Quartz crystal microbalance analysis confirmed binding of C3 to planar DMPC surfaces. In addition, we demonstrated that DMPC liposomes bound to or were phagocytized by granulocytes in a complement-dependent manner, as evidenced by the use of complement inhibitors. In summary, we have shown that C3 is activated both by convertase-dependent cleavage, preferentially in the fluid phase, by mechanisms which are not well elucidated, and also by contact activation into C3(H2O) on the DMPC surface. In particular, this contact activation has implications for the therapeutic regulation of complement activation during liposome treatment. (C) 2013 Elsevier Ltd. All rights reserved.
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