| 1. |
- Aisenbrey, Christopher, et al.
(author)
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Helix orientations in membrane-associated Bcl-XL determined by 15N-solid-state NMR spectroscopy
- 2007
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In: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 37:1, s. 71-80
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Journal article (peer-reviewed)abstract
- Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-xL is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-xL associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-xL its membrane interactions are weak. In the presence of membranes Bcl-xL can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-xL hydrophobic binding pocket becomes accessible for protein–protein interactions whilst the structure of the N-terminal region remains intact.
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| 2. |
- Aisenbrey, Christopher, et al.
(author)
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How is protein aggregation in amyloidogenic diseases modulated by biological membranes?
- 2008
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In: European Biophysics Journal. - : SpringerLink. - 0175-7571 .- 1432-1017. ; 37:3, s. 247-55
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Journal article (peer-reviewed)abstract
- The fate of proteins with amyloidogenic properties depends critically on their immediate biochemical environment. However, the role of biological interfaces such as membrane surfaces, as promoters of pathological aggregation of amyloidogenic proteins, is rarely studied and only established for the amyloid-β protein (Aβ) involved in Alzheimer’s disease, and α-synuclein in Parkinsonism. The occurrence of binding and misfolding of these proteins on membrane surfaces, is poorly understood, not at least due to the two-dimensional character of this event. Clearly, the nature of the folding pathway for Aβ protein adsorbed upon two-dimensional aggregation templates, must be fundamentally different from the three-dimensional situation in solution. Here, we summarize the current research and focus on the function of membrane interfaces as aggregation templates for amyloidogenic proteins (and even prionic ones). One major aspect will be the relationship between membrane properties and protein association and the consequences for amyloidogenic products. The other focus will be on a general understanding of protein folding pathways on two-dimensional templates on a molecular level. Finally, we will demonstrate the potential importance of membrane-mediated aggregation for non-amphiphatic soluble amyloidogenic proteins, by using the SOD1 protein involved in the amyotrophic lateral sclerosis syndrome.
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| 3. |
- Aisenbrey, Christopher, et al.
(author)
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Macromolecular Crowding at Membrane Interfaces : Adsorption and Alignment of Membrane Peptides
- 2008
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In: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 375, s. 376-385
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Journal article (peer-reviewed)abstract
- Association of proteins to cellular membranes is involved in various biological processes. Various theoretical models have been developed to describe this adsorption mechanism, commonly implying the concept of an ideal solution. However, due to the two-dimensional character of membrane surfaces intermolecular interactions between the adsorbed molecules become important. Therefore previously adsorbed molecules can influence the adsorption behavior of additional protein molecules and their membrane-associated structure. Using the model peptide LAH4, which upon membrane-adsorption can adopt a transmembrane as well as an in-planar configuration, we carried out a systematic study of the correlation between the peptide concentration in the membrane and the topology of this membrane-associated polypeptide. We could describe the observed binding behavior by establishing a concept, which includes intermolecular interactions in terms of a scaled particle theory.High surface concentration of the peptide shifts the molecules from an in-planar into a transmembrane conformation, a process driven by the reduction of occupied surface area per molecule. In a cellular context, the crowding-dependent alignment might provide a molecular switch for a cell to sense and control its membrane occupancy. Furthermore, crowding might have pronounced effects on biological events, such as the cooperative behavior of antimicrobial peptides and the membrane triggered aggregation of amyloidogenic peptides.
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| 4. |
- Aisenbrey, Christopher, et al.
(author)
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SOD1 associates to membranes in its folded apo-state
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Other publication (pop. science, debate, etc.)abstract
- Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease accompanied by misfolding and intracellular deposition of superoxide dismutase 1 (SOD1). Although the molecular details behind this misfolding process are yet poorly understood, increasing evidence suggest that SOD1 is most susceptible to misfolding in its metal-free and relatively unstable apo-state. Here, we addressed the question, if misfolding and aggregation of SOD1 involves erroneous interactions with membranes as has been implicated for the Aβ peptide in Alzheimers disease. To examine this possibility we subjected various apo SOD1 variants to the presence of different membrane systems. The results reveal that wild type apoSOD1 but to less extent destabilized ALS mutations interact with charged vesicles under physiologically relevant conditions, thereby acquiring pronounced helical structural features. As the data further show, the protein binds to the membranes by an electrostatically driven mechanism, which requires a folded apo-state conformation and a negative membrane surface potential. Unfolded SOD1 molecules show no appreciable affinity to the membrane surfaces yielding a correlation between increased stability, i. e. occupancy of folded molecules and extend of membrane association. Since this trend opposes the correlation between decreased SOD1 stability and progression of neural damage, the results suggest that membrane association is not part of the ALS mechanism. An explanation could be that the observed membrane association of apo SOD1 is reversible and does not ‘bleed out’ in irreversible aggregation as observed for other precursors of protein-misfolding diseases.
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| 5. |
- Aisenbrey, Christopher, et al.
(author)
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Specific Isotope Labeling of Colicin E1 and B Channel Domains For Membrane Topological Analysis by Oriented Solid-State NMR Spectroscopy
- 2008
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In: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 9:6, s. 944-951
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Journal article (peer-reviewed)abstract
- An approach is presented to selectively label the methionines of the colicin E1 and B channel domains, each about 200 residues in size, and use them for oriented solid-state NMR investigations. By combining site-directed mutagenesis, bacterial overexpression in a methionine auxotroph E. coli strain and biochemical purification, quantitative amounts of the proteins for NMR structural investigations were obtained. The proteins were selectively labeled with 15N at only one, or at a few, selected sites. Multidimensional heteronuclear correlation high-resolution NMR spectroscopy and mass spectrometry were used to monitor the quality of isotopic labeling. Thereafter the proteins were reconstituted into oriented phospholipid bilayers and investigated by proton-decoupled 15N solid-state NMR spectroscopy. The colicin E1 thermolytic fragment that carries a single 15N methionine within its hydrophobic helix 9 region exhibited 15N resonances that are characteristic of helices that are oriented predominantly parallel to the membrane surface at low temperature, and a variety of alignments and conformations at room temperature. This suggests that the protein can adopt both umbrella and pen-knife conformations.
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| 6. |
- Aisenbrey, Christopher, et al.
(author)
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Structure and dynamics of membrane-associated ICP47, a viral inhibitor of the MHC I antigen-processing machinery
- 2006
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In: The Journal of Biological Chemistry. - 0021-9258. ; 281:41, s. 30365-72
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Journal article (peer-reviewed)abstract
- To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.
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| 7. |
- Aisenbrey, Christopher, et al.
(author)
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Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy
- 2007
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In: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 36:4-5, s. 451-60
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Journal article (peer-reviewed)abstract
- Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the 15N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the 2H solid-state NMR spectra acquired from peptides labelled with 3,3,3-2H3-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the Cα–Cβ bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu1–27 and Influenza A M222–46 are shown. Whereas the 15N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed.
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| 8. |
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| 9. |
- Bugaytsova, Jeanna, et al.
(author)
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pH regulated H. pylori adherence : implications for persistent infection and disease
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Other publication (other academic/artistic)abstract
- Helicobacter pylori’s BabA adhesin binds strongly to gastric mucosal ABH/Leb glycans on the stomach epithelium and overlying mucus, materials continuously shed into the acidic gastric lumen. Here we report that this binding is acid labile, acid inactivation is fully reversible; and acid lability profiles vary with BabA sequence and correlate with disease patterns. Isogenic H. pylori strains from the gastric antrum and more acidic corpus were identified that differed in acid lability of receptor binding and in sequence near BabA’s carbohydrate binding domain. We propose that reversible acid inactivation of receptor binding helps H. pylori avoid clearance by mucosal shedding, and that strain differences in acid lability affect tissue tropism and the spectrum of associated gastric diseases.
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| 10. |
- Byström, Roberth, 1971-, et al.
(author)
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Disordered proteins : Biological membranes as two-dimensional aggregation matrices
- 2008
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In: Cell Biochemistry and Biophysics. - : Springer Science and Business Media LLC. - 1085-9195 .- 1559-0283. ; 52:3, s. 175-189
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Research review (peer-reviewed)abstract
- Aberrant folded proteins and peptides are hallmarks of amyloidogenic diseases. However, the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, despite intense efforts to establish a general molecular description of their behavior. Clearly, the fate of these proteins is ultimately linked to their immediate biochemical environment in vivo. In this review, we focus on the role of biological membranes, reactive interfaces that not only affect the conformational stability of amyloidogenic proteins, but also their aggregation rates and, probably, their toxicity. We first provide an overview of recent work, starting with findings regarding the amphiphatic amyloid-β protein (Aβ), which give evidence that membranes can directly promote aggregation, and that the effectiveness in this process can be related to the presence of specific neuronal ganglioside lipids. In addition, we discuss the implications of recent research (medin as an detailed example) regarding putative roles of membranes in the misfolding behavior of soluble, non-amphiphatic proteins, which are attracting increasing interest. The potential role of membranes in exerting the toxic action of misfolded proteins will also be highlighted in a molecular context. In this review, we discuss novel NMR-based approaches for exploring membrane–protein interactions, and findings obtained using them, which we use to develop a molecular concept to describe membrane-mediated protein misfolding as a quasi-two-dimensional process rather than a three-dimensional event in a biochemical environment. The aim of the review is to provide researchers with a general understanding of the involvement of membranes in folding/misfolding processes in vivo, which might be quite universal and important for future research concerning amyloidogenic and misfolding proteins, and possible ways to prevent their toxic actions.
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| 11. |
- Byström, Roberth, 1971-, et al.
(author)
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Electrostatic interactions between negatively charged phospolipid membranes and SOD1 protein : Effect of charge changing fALS mutations
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Other publication (pop. science, debate, etc.)abstract
- The neurodegenerative disease amyotrophic lateral sclerosis (ALS) is closely connected to single site mutations of the Cu/Zn superoxide dismutase (SOD1) protein, whose pathological conversion into misfolded aggregates is a hallmark of ALS. To explore the impact of protein net charge changing ALS relevant SOD1 mutations on their ability to interact with neuronal membranes and the consequences for their folding behaviour, we studied by circular dichroism the conformational changes of the SOD1pWT, SOD1N86D and SOD1N86K species in their apo-state in the presence of increasing amounts of negatively charged lipid bilayers.. The results clearly indicate an electrostatically driven association process, where the association event induces a pronounced increase in the helical character of the pWT and the N86D species, characterized by long patient survival times. To the opposite, the charge reducing N86K mutation shows more pronounced β-like features in the presence of membranes in comparison to the other two species; an observation which most likely reflects its reduced stability in its apo-state in combination with a very fast ALS progression.
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| 12. |
- Nordlund, Anna, et al.
(author)
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Functional features cause misfolding of the ALS-provoking enzyme SOD1
- 2009
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In: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 106:24, s. 9667-9672
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Journal article (peer-reviewed)abstract
- The structural integrity of the ubiquitous enzyme superoxide dismutase (SOD1) relies critically on the correct coordination of Cu and Zn. Loss of these cofactors not only promotes SOD1 aggregation in vitro but also seems to be a key prerequisite for pathogenic misfolding in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We examine here the consequences of Zn2+ loss by selectively removing the Zn site, which has been implicated as the main modulator of SOD1 stability and disease competence. After Zn-site removal, the remaining Cu ligands can coordinate a non-native Zn2+ ion with mu M affinity in the denatured state, and then retain this ion throughout the folding reaction. Without the restriction of a metallated Zn site, however, the Cu ligands fail to correctly coordinate the nonnative Zn2+ ion: Trapping of a water molecule causes H48 to change rotamer and swing outwards. The misligation is sterically incompatible with the native structure. As a consequence, SOD1 unfolds locally and interacts with neighboring molecules in the crystal lattice. The findings point to a critical role for the native Zn site in controlling SOD1 misfolding, and show that even subtle changes of the metal-loading sequence can render the wild-type protein the same structural properties as ALS-provoking mutations. This frustrated character of the SOD1 molecule seems to arise from a compromise between optimization of functional and structural features.
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| 13. |
- Åberg, Anna, et al.
(author)
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Helicobacter pylori adapts to chronic infection and gastric disease via ph-responsive baba-mediated adherence
- 2017
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In: Cell Host and Microbe. - : Elsevier BV. - 1931-3128 .- 1934-6069. ; 21:3, s. 376-389
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Journal article (peer-reviewed)abstract
- The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.
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