| 1. |
- Andrésen, Cecilia
(författare)
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Protein Structure and Interaction in Health and Disease
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis focuses on protein structure, dynamics and interaction and their relation to human disease. In particular, the biophysical and structural properties of both well-ordered and partially disordered proteins are studied using a range of biophysical techniques such as circular dichroism spectroscopy, fluorescence spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. Pseudomonas aeruginosa is a human pathogen due to its multidrug resistance (MDR) caused by overexpression of efflux pump systems. This thesis describes how MDR mutations within the MexR repressor of the MexAB-OprM system reduce the DNA affinity by altering its stability with maintained structure. The oncogenic protein c-Myc is involved in many essential biological functions such as cell proliferation, differentiation and apoptosis and is also highly associated with several forms of human cancers, and where the N-terminal domain is regulated by a plethora of protein interactions. In this thesis the intrinsically disordered N-terminal part of c-Myc and its interactions with the proteins Bin1 and TBP are described. Myc binds Bin1 with maintained disorder in a multivalent manner, which may explain why the onco-protein can interact with such a wide range of binding partners. A similarly dynamic interaction is observed for Myc with the TATA-binding protein (TBP). The essential human multidomain glutaredoxin Grx3 is associated with several biological functions such as redox signaling, proliferation and signal transduction. We have solved the structure and analyzed the dynamic properties in the ps-ns and ms time scale for the two N-terminal domains, providing a platform for further analysis of the Grx3 protein and its interactions. Taken together, this thesis emphasizes the importance of joint structural, biophysical and dynamic studies to better understand protein function in health and disease.
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| 2. |
- Ariöz, Candan, 1983-, et al.
(författare)
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Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:33, s. 5533-5544
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Tidskriftsartikel (refereegranskat)abstract
- Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.
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| 3. |
- Björnerås, Johannes, et al.
(författare)
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Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the kappa opioid receptor using a soluble protein scaffold
- 2014
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:3, s. 814-824
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Tidskriftsartikel (refereegranskat)abstract
- The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the kappa opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble beta-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.
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| 4. |
- Björnerås, Johannes, 1982-
(författare)
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Dynorphin A – Interactions with receptors and the membrane bilayer
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis concerns the dynorphin neuropeptides, and dynorphin A (DynA) in particular. DynA belongs to the wider class of typical opioid peptides that, together with the opioid receptors, a four-membered family of GPCR membrane proteins, form the opioid system. This biological system is involved or implicated in several physiological processes such as analgesia, addiction and depression, and effects caused by DynA through this system, mainly through interaction with the kappa subtype of the opioid receptors (KOR), are called the opioid effects. In addition to this, non-opioid routes of action for DynA have been proposed, and earlier studies have shown that direct membrane interaction is likely to contribute to these non-opioid effects. The results discussed here fall into either of two categories; the interaction between DynA and a fragment of KOR, and the direct lipid interaction of DynA and two variant peptides.For the receptor interaction case, DynA most likely causes its physiological effects through binding its N-terminal into a transmembrane site of the receptor protein, while the extracellular regions of the protein, in particular the extracellular loop II (EL2), have been shown to be important for modulating the selectivity of KOR for DynA. Here we have focussed on the EL2, and show the feasibility of transferring this sequence into a soluble protein scaffold. Studies, predominantly by nuclear magnetic resonance (NMR) spectroscopy, of EL2 in this new environment show that the segment has the conformational freedom expected of a disordered loop sequence, while the scaffold keeps its native beta-barrel fold. NMR chemical shift and paramagnetic resonance enhancement experiments show that DynA binds with high specificity to EL2 with a dissociation constant of approximately 30 micro Molar, while binding to the free EL2 peptide is an order of magnitude weaker. The strength of these interactions are reasonable for a receptor recognition event. No binding to the naked scaffold protein is observed.In the second project, the molecules of interest were two DynA peptide variants recently found in humans and linked to a neurological disorder. Previously published reports from our group and collaborators pointed at very different membrane-perturbing properties for the two variants, and here we present the results of a follow-up study, where the variants R6W-DynA and L5S-DynA were studied by NMR and circular dichroism (CD) spectroscopy in solutions of fast-tumbling phospholipid bicelles, and compared with wild type DynA. Our results show that R6W-DynA interacts slightly stronger with lipids compared to wild type DynA, and much stronger compared to L5S-DynA, in terms of bicelle association, penetration and structure induction. These results are helpful for explaining the differences in toxicity, membrane perturbation and relationship to disease, between the studied neuropeptides.
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| 5. |
- Björnerås, Johannes, et al.
(författare)
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Membrane Interaction of Disease-Related Dynorphin A Variants
- 2013
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:24, s. 4157-4167
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Tidskriftsartikel (refereegranskat)abstract
- The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small a-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (similar to 90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (similar to 50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.
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| 6. |
- Björnerås, Johannes, 1982-
(författare)
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The opioid peptide dynorphin A : Biophysical studies of peptide–receptor and peptide–membrane interactions
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA.The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event.With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge.Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic.
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| 7. |
- Dinic, Jelena, et al.
(författare)
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Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing
- 2011
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1808:1, s. 298-306
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Tidskriftsartikel (refereegranskat)abstract
- Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.
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| 8. |
- Gräslund, Astrid, et al.
(författare)
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Testing membrane interactions of CPPs
- 2011
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Ingår i: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1940-6029. ; 683, s. 33-40
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Tidskriftsartikel (refereegranskat)abstract
- The chapter deals with some biophysical methods used for investigating CPP-induced changes in membrane properties by spectroscopy methods such as fluorescence or NMR and methods used for probing CPP-induced leakage in membranes. Some useful model systems for biomembranes are described. These include large unilamellar phospholipid vesicles (LUVs) of well-defined size (diameter typically 100 nm). A protocol for the preparation of such vesicles is included. The leakage studies make use of LUVs with entrapped dye molecules. The NMR studies make use of mixed micelles (bicelles) as a membrane mimetic system, which can be oriented in the magnetic field of the spectrometer.
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| 9. |
- Jornvall, Hans, et al.
(författare)
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Oligomerization and insulin interactions of proinsulin C-peptide : Threefold relationships to properties of insulin
- 2010
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 391:3, s. 1561-1566
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Tidskriftsartikel (refereegranskat)abstract
- Three principally different sites of action have been reported for proinsulin C-peptide, at surface-mediated, intracellular, and extracellular locations. Following up on the latter, we now find that (i) mass spectrometric analyses reveal the presence of the C-peptide monomer in apparent equilibrium with a low-yield set of oligomers in weakly acidic or basic aqueous solutions, even at low peptide concentrations (sub-mu M). It further shows not only C-peptide to interact with insulin oligomers (known before), but also the other way around. (ii) Polyacrylamide gel electrophoresis of C-peptide shows detectable oligomers upon Western blotting. Formation of thioflavin T positive material was also detected. (iii) Cleavage patterns of analogues are compatible with C-peptide as a substrate of insulin degrading enzyme. Combined, the results demonstrate three links with insulin properties, in a manner reminiscent of amyloidogenic peptides and their chaperons in other systems. If so, peripheral C-peptide/insulin interactions, absolute amounts of both peptides and their ratios may be relevant to consider in diabetic and associated diseases.
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| 10. |
- Lind, Jesper, et al.
(författare)
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Structural features of proinsulin C-peptide oligomeric and amyloid states
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:18, s. 3759-68
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Tidskriftsartikel (refereegranskat)abstract
- The formation and structure of proinsulin C-peptide oligomers has been investigated by PAGE, NMR spectroscopy and dynamic light scattering. The results obtained show that C-peptide forms oligomers of different sizes, and that their formation and size distribution is altered by salt and divalent metal ions, which indicates that the aggregation process is mediated by electrostatic interactions. It is further demonstrated that the size distribution of the C-peptide oligomers, in agreement with previous studies, is altered by insulin, which supports a physiologically relevant interaction between these two peptides. A small fraction of oligomers has previously been suggested to be in equilibrium with a dominant fraction of soluble monomers, and this pattern also is observed in the present study. The addition of modest amounts of sodium dodecyl sulphate at low pH increases the relative amount of oligomers, and this effect was used to investigate the details of both oligomer formation and structure by a combination of biophysical techniques. The structural properties of the SDS-induced oligomers, as obtained by thioflavin T fluorescence, CD spectroscopy and IR spectroscopy, demonstrate that soluble aggregates are predominantly in β-sheet conformation, and that the oligomerization process shows characteristic features of amyloid formation. The formation of large, insoluble, β-sheet amyloid-like structures will alter the equilibrium between monomeric C-peptide and oligomers. This leads to the conclusion that the oligomerization of C-peptide may be relevant also at low concentrations.
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