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| 5. |
- Aisenbrey, Christopher, et al.
(författare)
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Helix orientations in membrane-associated Bcl-XL determined by 15N-solid-state NMR spectroscopy
- 2007
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 37:1, s. 71-80
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Aisenbrey, Christopher, et al.
(författare)
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How is protein aggregation in amyloidogenic diseases modulated by biological membranes?
- 2008
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Ingår i: European Biophysics Journal. - : SpringerLink. - 0175-7571 .- 1432-1017. ; 37:3, s. 247-55
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Aisenbrey, Christopher, et al.
(författare)
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Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy
- 2007
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 36:4-5, s. 451-60
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Tidskriftsartikel (refereegranskat)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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- Armstrong, Clare L., et al.
(författare)
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Effect of cholesterol on the lateral nanoscale dynamics of fluid membranes
- 2012
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 41:10, s. 901-913
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Tidskriftsartikel (refereegranskat)abstract
- Inelastic neutron scattering was used to study the effect of 5 and 40 mol% cholesterol on the lateral nanoscale dynamics of phospholipid membranes. By measuring the excitation spectrum at several lateral q (||) values (up to q (||) = 3 (-1)), complete dispersion curves were determined of gel, fluid and liquid-ordered phase bilayers. The inclusion of cholesterol had a distinct effect on the collective dynamics of the bilayer's hydrocarbon chains; specifically, we observed a pronounced stiffening of the membranes on the nanometer length scale in both gel and fluid bilayers, even though they were experiencing a higher degree of molecular disorder. Also, for the first time we determined the nanoscale dynamics in the high-cholesterol liquid-ordered phase of bilayers containing cholesterol. Namely, this phase appears to be "softer" than fluid bilayers, but better ordered than bilayers in the gel phase.
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| 18. |
- Bally, Marta
(författare)
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Regulating the dynamic interactions between herpes simplex viruses and cell -surface glycosaminoglycans
- 2019
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Ingår i: European Biophysics Journal. - : Springer. - 0175-7571 .- 1432-1017. ; 48, s. S41-S41
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Virus entry is a complex dynamic multistep process requiring a series of fine-tuned events mediating virus diffusion through the glycocalyx, its attachment to the cell membrane and lateral diffusion to the point of entry. A number of enveloped viruses, including herpes simplex viruses (HSV) attach to susceptible host cells via interaction between their glycoproteins and cell-surface glycosaminoglycans (GAGs). In our work, we study the molecular and physical mechanisms modulating HSV binding, diffusion and release from cell-surface glycosaminoglycans. Using single virus tracking in combination with either in vitro minimal models of the cell surface or live cell microscopy, we gain insights into the modulatory function of protein glycosylation (the presence of mucin-like regions on viral glycoproteins) and interrogate the role of GAG sulfation in the process. We show that mucin-like regions found on the glycoproteins of HSV-1 and HSV-2 play an important role in modulating the interaction, an observation further supported by cell experiments. We further show that the diffusion of virions on the surface depends on the type of GAGs and their degree of sulfation. Taken together, our research contributes to a better understanding of the mechanisms underlying the interaction between a virus and the surface of its host. Such insights will without doubt facilitate the design of more efficient antiviral drugs or vaccines.
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| 22. |
- Bernson, David, 1986, et al.
(författare)
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Amyloid formation of bovine insulin is retarded in moderately acidic pH and by addition of short-chain alcohols
- 2020
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 1432-1017 .- 0175-7571. ; 49:2, s. 145-153
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Tidskriftsartikel (refereegranskat)abstract
- Protein aggregation and amyloid formation are associated with multiple human diseases, but are also a problem in protein production. Understanding how aggregation can be modulated is therefore of importance in both medical and industrial contexts. We have used bovine insulin as a model protein to explore how amyloid formation is affected by buffer pH and by the addition of short-chain alcohols. We find that bovine insulin forms amyloid fibrils, albeit with different rates and resulting fibril morphologies, across a wide pH range (2-7). At pH 4.0, bovine insulin displayed relatively low aggregation propensity in combination with high solubility; this condition was therefore chosen as basis for further exploration of how bovine insulin's native state can be stabilized in the presence of short-chain alcohols that are relevant because of their common use as eluents in industrial-scale chromatography purification. We found that ethanol and isopropanol are efficient modulators of bovine insulin aggregation, providing a three to four times retardation of the aggregation kinetics at 30-35% (vol/vol) concentration; we attribute this to the formation of oligomers, which we detected by AFM. We discuss this effect in terms of reduced solvent polarity and show, by circular dichroism recordings, that a concomitant change in alpha-helical packing of the insulin monomer occurs in ethanol. Our results extend current knowledge of how insulin aggregates, and may, although bovine insulin serves as a simplistic model, provide insights into how buffers and additives can be fine-tuned in industrial production of proteins in general and pharmaceutical insulin in particular.
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| 23. |
- Björnham, Oscar, 1976-, et al.
(författare)
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Methods and error estimations of uncertainties in single-molecule dynamic force spectroscopy
- 2009
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 38:7, s. 911-922
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Tidskriftsartikel (refereegranskat)abstract
- In dynamic force spectroscopy, access to the characteristic parameters of single molecular bonds requires non-trivial measurements and data processing as the rupture forces are found not only to be distributed over a wide range, but are also dependent on the loading rate. The choice of measurement procedure and data processing methods has a considerable impact on the accuracy and precision of the final results. We analyse, by means of numerical simulations, methods to minimize and assess the magnitude of the expected errors for different combinations of experimental and evaluation methods. It was found that the choice of fitting function is crucial to extract correct parameter values. Applying a Gaussian function, which is a common practice, is equivalent to introducing a systematic error, and leads to a consequent overestimation of the thermal off-rate by more than 30%. We found that the precision of the bond length and the thermal off-rate, in presence of unbiased noise, were improved by reducing the number of loading rates for a given number of measurements. Finally, the results suggest that the minimum number of measurements needed to obtain the bond strength, with acceptable precision, exceeds the common number of ~100 reported in literature.
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| 24. |
- Björnham, Oscar, 1976-, et al.
(författare)
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Modeling of the elongation and retraction of Escherichia coli P pili under strain by Monte Carlo simulations
- 2008
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 37:4, s. 381-391
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Tidskriftsartikel (refereegranskat)abstract
- P pili are fimbrial adhesion organelles expressed by uropathogenic Escherichia coli in the upper urinary tract. They constitute a stiff helix-like polymer consisting of a number of subunits joined by head-to-tail bonds. The elongation and retraction properties of individual P pili exposed to strain have been modeled by Monte Carlo (MC) simulations. The simulation model is based upon a three-state energy landscape that deforms under an applied force. Bond opening and closure are modeled by Bells theory while the elongation of the linearized part of the pilus is described by a worm-like chain model. The simulations are compared with measurements made by force measuring optical tweezers. It was found that the simulations can reproduce pili elongation as well as retraction, under both equilibrium and dynamic conditions, including entropic effects. It is shown that the simulations allow for an assessment of various model parameters, e.g. the unfolding force, energy barrier heights, and various distances in the energy landscape, including their stochastic spread that analytical models are unable to do. The results demonstrate that MC simulations are useful to model elongation and retraction properties of P pili, and therefore presumably also other types of pili, exposed to strain and/or stress. MC simulations are particularly suited for description of helix-like pili since these have an intricate self-regulating mechanical elongation behavior that makes analytical descriptions non-trivial when dynamic processes are studied, or if additional interactions in the rod or the behavior of the adhesion tip needs to be modeled.
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| 25. |
- Björnham, Oscar, 1976-, et al.
(författare)
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Physical properties of the specific PapG–galabiose binding in E. coli P pili-mediated adhesion
- 2009
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Ingår i: European Biophysics Journal. - New York : Springer. - 0175-7571 .- 1432-1017. ; 38:2, s. 245-254
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Tidskriftsartikel (refereegranskat)abstract
- Detailed analyses of the mechanisms thatmediate binding of the uropathogenic Escherichia coli tohost cells are essential, as attachment is a prerequisite forthe subsequent infection process. We explore, by means offorce measuring optical tweezers, the interaction betweenthe galabiose receptor and the adhesin PapG expressed byP pili on single bacterial cells. Two variants of dynamicforce spectroscopy were applied based on constant andnon-linear loading force. The specific PapG–galabiosebinding showed typical slip-bond behaviour in the forceinterval (30–100 pN) set by the pilus intrinsic biomechanicalproperties. Moreover, it was found that the bondhas a thermodynamic off-rate and a bond length of2.6×10-3 s-1 and 5.0 Å , respectively. Consequently, thePapG–galabiose complex is significantly stronger thanthe internal bonds in the P pilus structure that stabilizes thehelical chain-like macromolecule. This finding suggeststhat the specific binding is strong enough to enable the Ppili rod to unfold when subjected to strong shear forces inthe urinary tract. The unfolding process of the P pili rodpromotes the formation of strong multipili interaction,which is important for the bacterium to maintain attachmentto the host cells.
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| 26. |
- Björnham, Oscar, et al.
(författare)
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Theory for nonlinear dynamic force spectroscopy
- 2017
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 46:3, s. 225-233
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Tidskriftsartikel (refereegranskat)abstract
- Dynamic force spectroscopy (DFS) is an experimental technique that is commonly used to assess information on the strength, energy landscape, and lifetime of noncovalent bio-molecular interactions. DFS traditionally requires an applied force that increases linearly with time so that the bio-complex under investigation is exposed to a constant loading rate. However, tethers or polymers can modulate the applied force in a nonlinear manner. For example, bacterial adhesion pili and polymers with worm-like chain properties are structures that show nonlinear force responses. In these situations, the theory for traditional DFS cannot be readily applied. In this work, we expand the theory for DFS to also include nonlinear external forces while still maintaining compatibility with the linear DFS theory. To validate the theory, we modeled a bio- complex expressed on a stiff, an elastic, and a worm-like chain polymer, using Monte Carlo methods, and assessed the corresponding rupture force spectra. It was found that the nonlinear DFS (NLDFS) theory correctly predicted the numerical results. We also present a protocol suggesting an experimental approach and analysis method of the data to estimate the bond length and the thermal off-rate.
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| 27. |
- Blobel, Jascha, et al.
(författare)
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Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins
- 2011
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 40:12, s. 1327-1338
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Tidskriftsartikel (refereegranskat)abstract
- Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.
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| 31. |
- Castelain, Mickaël, et al.
(författare)
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Fast uncoiling kinetics of F1C pili expressed by uropathogenic Escherichia coli are revealed on a single pilus level using force-measuring optical tweezers
- 2011
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 40:3, s. 305-316
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Tidskriftsartikel (refereegranskat)abstract
- Uropathogenic Escherichia coli (UPEC) expressvarious kinds of organelles, so-called pili or fimbriae, thatmediate adhesion to host tissue in the urinary tract throughspecific receptor-adhesin interactions. The biomechanicalproperties of these pili have been considered important forthe ability of bacteria to withstand shear forces from rinsingurine flows. Force-measuring optical tweezers have beenused to characterize individual organelles of F1C typeexpressed by UPEC bacteria with respect to such properties.Qualitatively, the force-versus-elongation response wasfound to be similar to that of other types of helix-like piliexpressed by UPEC, i.e., type 1, P, and S, with force-inducedelongation in three regions, one of which represents theimportant uncoiling mechanism of the helix-like quaternarystructure. Quantitatively, the steady-state uncoiling forcewas assessed as 26.4 ±1.4 pN, which is similar to those ofother pili (which range from 21 pN for SI to 30 pN for type 1).The corner velocity for dynamic response (1,400 nm/s) wasfound to be larger than those of the other pili (400–700 nm/sfor S and P pili, and 6 nm/s for type 1). The kinetics werefound to be faster, with a thermal opening rate of 17 Hz, afew times higher than S and P pili, and three orders ofmagnitude higher than type 1. These data suggest that F1Cpili are, like P and S pili, evolutionarily selected to primarilywithstand the conditions expressed in the upper urinary tract.
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| 33. |
- Castelain, Mickaël, et al.
(författare)
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Unfolding and refolding properties of S pili on extraintestinal pathogenic Escherichia coli
- 2010
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 39:8, s. 1105-1115
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Tidskriftsartikel (refereegranskat)abstract
- S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (S(II)) and believed to play a role in ascending urinary tract infections (S(I)). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of S(II) and S(I) pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for S(II) pili and 21 pN for S(I) pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX (II)) indicated that absence of the sfaX (II) gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of S(I) are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.
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| 38. |
- Conca, Dario Valter, et al.
(författare)
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The role of membrane complexity in the early entry stages of SARS-CoV-2 variants
- 2023
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Ingår i: European Biophysics Journal. - 1432-1017 .- 0175-7571. ; 52:SUPPL 1, s. S176-S176
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The highest density of mutations in SARS-CoV-2 variants is located on the spike glycoprotein (S), which is responsible for receptor ACE2 engagement. This suggests that SARS-CoV-2 is evolving to optimize viral entry. Several molecular studies report differences in the affinity between isolated S and ACE2 among variants. However, overall ACE2 affinity poorly correlates with the increased infectivity of recent variants. We address this discrepancy by considering the virus interaction with the whole plasma membrane and study the role of avidity and membrane complexity in modulating virus-host binding. To this end, we employ an in-vitro model system combining single-particle tracking and native supported lipid bilayers (nSLBs) made from lung epithelial cells. As virion mimics, we developed S-decorated liposomes that allow for direct comparison between variants and BSL-1 handling. Sliposome interaction with nSLBs showed a significant increase in avidity for Omicron compared to Delta and Wuhan strains. Further, using single-molecule force spectroscopy, we reveal a higher affinity for Omicron and Delta S to sensor immobilise heparan sulfate (HS). Our results indicate a shift in the variants’ attachment strategy towards more efficient use of coreceptors and the role of HS as an initial docking site that facilitates virus accumulation at the membrane and ACE2 engagement.
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