| 1. |
- Andersson, Håkan S., et al.
(författare)
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The alpha-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance
- 2012
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Ingår i: Amino Acids. - : Springer Science and Business Media LLC. - 0939-4451 .- 1438-2199. ; 43:4, s. 1471-1483
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Tidskriftsartikel (refereegranskat)abstract
- Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the alpha-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg(7) or Glu(15) residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg(7) and Glu(15) substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic alpha-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical alpha-defensin salt-bridge facilitates adoption of the characteristic alpha-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.
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| 2. |
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| 3. |
- Bengtsson, Elina, et al.
(författare)
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Actomyosin Interactions and Different Structural States of Actin Filaments
- 2013
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 104:2 Suppl. 1, s. 480A-481A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The persistence length (LP) of a polymer is proportional to its flexural rigidity and quantifies the decay length of its tangent angle (for a polymer freely suspended in solution). Further, it has been suggested that the decay length for the sliding direction of heavy meromyosin (HMM) propelled actin filaments in the in vitro motility assay (IVMA) is quantitatively identical to Lp of the free leading filament end. On this assumption we measured LP under different conditions to address a hypothesis that the actin filament exists in different metastable conformations, each characterized by a different flexural rigidity. The following values for Lp (mean 5 95 % confidence limits) were obtained: 1. with phalloidin (Ph) in solution: 12.61 5 0.65 mm (N=809). 2. without Phin solution: 9.07 5 1.06 mm (N=811), 3. with Ph and HMM in solution (rigor):10.21 5 0.75 mm (N=429), 4. without Ph (from IVMA paths; 1 mM MgATP):10.0850.66 mm (N=309), 5. with Ph, IVMA (1 mM MgATP): 11.41 5 0.57 mm (N=243), 6. with Ph, IVMA, 0.05 mM MgATP: 6.30 5 0.27 mm (N=383) and 7. without Ph, IVMA, 0.02-0.05 mM MgATP: 5.33 5 0.37 mm (N=161). The re-sults are consistent with different actin filament states where one is stabilized by phalloidin and one is favored by HMM binding and the absence of Ph. Effects of HMM are consistent with a possible role of the structural state of actin filaments in effective actomyosin motility. The very low LP found for IVMA at low [MgATP] (6-7) may reflect the presence of an actin filament state populated at low average cross-bridge strains, possibly with MgADP at the active site. Alternatively, it may be due to sideways forces produced by increased number of HMM-actin interactions close to the leading filament end.
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| 4. |
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| 5. |
- Bengtsson, Elina, et al.
(författare)
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Altered Structural State of Actin Filaments Upon MYOSIN II Binding
- 2015
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 108:2 Suppl. 1, s. 299A-300A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The paths of actin filaments propelled over a heavy meromyosin (HMM) surface in the in vitro motility assay (IVMA) can statistically be described by a path persistence length (LPP) and has been hypothesized to be proportional to the flexural rigidity of the filaments. Here, we have studied the LPP at high (130 mM) ionic strength along with the persistence length of actin filaments in solution (LPS) to elucidate how HMM binding affects the flexural rigidity of actin filaments. Characterization and control of material properties, such as the path persistence length, is useful in engineered devices that takes advantages of the function of the muscle contractile proteins e.g. for biocomputation. It has been suggested that myosin binding reduces Lpp for phalloidin stabilizedact in filaments. This is consistent with the results presented here where the phalloidin stabilized actin filaments rigidity is reduced to the level of phalloidin free actin filaments in the IVMA. Further, reducing the MgATP concentration in the IVMA would increase the HMM head density along the actin filament hence making the effect of myosin binding more pronounced. A reduced [MgATP] from 1 mM to 0.02-0.05 mM did indeed reduce the LPP from 10-12 mm to 6-7 mm for both phalloidin-stabilized and phalloidin free actin filaments. Additionally, we found a negative correlation between the LPS and the [HMM]/actin ratio. However, this [HMM] dependent reduction observed in LPS was too small to account for the reduction in LPP seen with reduced [MgATP] in the IVMA. Monte-Carlo simulations and theoretical analysis revealed that the large reduction in LPP is consistent with the idea that every head attachment adds an extra angular displacement.(Support from EU-FP7-FET-ABACUS grant number 613044).
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| 6. |
- Bengtsson, Elina, et al.
(författare)
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Analysis of Flexural Rigidity of Actin Filaments Propelled by Surface Adsorbed Myosin Motors
- 2013
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Ingår i: Cytoskeleton. - : John Wiley & Sons. - 1949-3584 .- 1949-3592. ; 70:11, s. 718-728
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Tidskriftsartikel (refereegranskat)abstract
- Actin filaments are central components of the cytoskeleton and the contractile machinery of muscle. The filaments are known to exist in a range of conformational states presumably with different flexural rigidity and thereby different persistence lengths. Our results analyze the approaches proposed previously to measure the persistence length from the statistics of the winding paths of actin filaments that are propelled by surface-adsorbed myosin motor fragments in the in vitro motility assay. Our results suggest that the persistence length of heavy meromyosin propelled actin filaments can be estimated with high accuracy and reproducibility using this approach provided that: (1) the in vitro motility assay experiments are designed to prevent bias in filament sliding directions, (2) at least 200 independent filament paths are studied, (3) the ratio between the sliding distance between measurements and the camera pixel-size is between 4 and 12, (4) the sliding distances between measurements is less than 50% of the expected persistence length, and (5) an appropriate cut-off value is chosen to exclude abrupt large angular changes in sliding direction that are complications, e.g., due to the presence of rigor heads. If the above precautions are taken the described method should be a useful routine part of in vitro motility assays thus expanding the amount of information to be gained from these.
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| 7. |
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| 8. |
- Bengtsson, Elina, et al.
(författare)
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Myosin-Induced Gliding Patterns at Varied [MgATP] Unveil a Dynamic Actin Filament
- 2016
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 111:7, s. 1465-1477
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Tidskriftsartikel (refereegranskat)abstract
- Actin filaments have key roles in cell motility but are generally claimed to be passive interaction partners in actin-myosin -based motion generation. Here, we present evidence against this static view based on an altered myosin-induced actin filament gliding pattern in an in vitro motility assay at varied [MgATP]. The statistics that characterize the degree of meandering of the actin filament paths suggest that for [MgATP] >= 0.25 mM, the flexural rigidity of heavy meromyosin (HMM)-propelled actin filaments is similar (without phalloidin) or slightly lower (with phalloidin) than that of HMM-free filaments observed in solution without surface tethering. When [MgATP] was reduced to <= 0.1 mM, the actin filament paths in the in vitro motility assay became appreciably more winding in both the presence and absence of phalloidin. This effect of lowered [MgATP] was qualitatively different from that seen when HMM was mixed with ATP-insensitive, N-ethylmaleimide-treated HMM (NEM-HMM; 25-30%). In particular, the addition of NEM-HMM increased a non-Gaussian tail in the path curvature distribution as well as the number of events in which different parts of an actin filament followed different paths. These effects were the opposite of those observed with reduced [MgATP]. Theoretical modeling suggests a 30-40% lowered flexural rigidity of the actin filaments at [MgATP] <= 0.1 mM and local bending of the filament front upon each myosin head attachment. Overall, the results fit with appreciable structural changes in the actin filament during actomyosin-based motion generation, and modulation of the actin filament mechanical properties by the dominating chemomechanical actomyosin state.
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| 9. |
- Bengtsson, Elina, et al.
(författare)
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Winding Actin Filament Paths Provide Mechanistic Insights Into Actomyosin Function
- 2012
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 102:3 Suppl. 1, s. 146A-146A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The statistics of heavy meromyosin (HMM) driven actin filament paths in vitro, and thermal fluctuations of actin filaments suspended in a pseudo 2D-space in solution, can be described by the cosine correlation equation (CCE): = exp(-s/[2*Lp]). Here, q0) and qs) represent tangent angles at distance 0 and s, respectively from one filament end (in solution) or from the starting point of the path. The quantity Lp is the persistence length (proportional to flexural rigidity) of the filament/path. In vitro motility assay (IVMA) studies (27-29oC) were performed along with studies of actin filaments suspended between two cover-slips in solution. Fits to the CCE gave LP = 16.5 5 1.7 mm (mean 5 95 % confidence interval) and 11.1 5 0.6 mm for phalloidin stabilized filaments in solution and propelled by HMM, respectively. In contrast, phalloidin free actin filaments (NHS-rhodamine labeled) exhibited similar LP in solution 10.1 52.1 mm and during HMM propulsion (9.8 5 0.9 mm). The filament paths were modeled using a Monte-Carlo approach updating angular changes in sliding direction at short time intervals (dt) assuming 1. lateral displacements due to cross-bridge forces and 2. thermal fluctuations of the leading filament end. The results suggest that > 3nm average lateral displacement during each actomyosin interaction would reduce LP by > 30 % compared to that of filaments without HMM. The findings are consistent with the following ideas: 1. Actin filaments exist in two different flexural rigidity states, one favored by myosin binding and the other by phalloidin stabilization, 2. Changes in actin filament flexural rigidity is not required for motion generation. 3. The myosin cross-bridges produce minimal lateral movements (< 3 nm) during the power-stroke.
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| 10. |
- Edefell, Ellen, et al.
(författare)
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Digging deep into a GAC filter – Temporal and spatial profiling of adsorbed organic micropollutants
- 2022
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Ingår i: Water Research. - : Elsevier BV. - 0043-1354 .- 1879-2448. ; 218
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Tidskriftsartikel (refereegranskat)abstract
- A large pilot-scale granular activated carbon (GAC) filter was operated downstream in a full-scale wastewater treatment plant to remove organic micropollutants. To describe the spatial and temporal developments of micropollutant adsorption profiles in the GAC filter, micropollutants were extracted from GAC media taken at various filter depths and number of treated bed volumes. At a low number of treated bed volumes (2600 BVs), most micropollutants were adsorbed in the top layers of the filter. At increasing number of treated bed volumes (7300–15,500 BVs), the adsorption front for micropollutants progressed through the filter bed at varying rates, with sulfamethoxazole, fluconazole, and PFOS reaching the bottom layer before carbamazepine and other well-adsorbing micropollutants, such as propranolol and citalopram. Higher amounts of adsorbed micropollutants in the bottom layer of the filter bed resulted in decreased removal efficiencies in the treated wastewater. Mass estimations indicated biodegradation for certain micropollutants, such as naproxen, diclofenac, and sulfamethoxazole. A temporary increase in the concentration of the insecticide imidacloprid could be detected in the filter indicating that extraction of adsorbed micropollutants could provide an opportunity for backtracking of loading patterns.
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