| 1. |
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| 2. |
- Abelein, Axel, et al.
(författare)
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Hydrophobicity and conformational change as mechanistic determinants for nonspecific modulators of amyloid β self-assembly
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:1, s. 126-137
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Tidskriftsartikel (refereegranskat)abstract
- The link between many neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer's disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.
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| 3. |
- Abelein, Axel, et al.
(författare)
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Transient small molecule interactions kinetically modulate amyloid beta peptide self-assembly
- 2012
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 586:22, s. 3991-3995
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Tidskriftsartikel (refereegranskat)abstract
- Small organic molecules, like Congo red and lacmoid, have been shown to modulate the self-assembly of the amyloid beta peptide (A beta). Here, we show that A beta forms NMR invisible non-toxic co-aggregates together with lacmoid as well as Congo red. We find that the interaction involves two distinct kinetic processes and at every given time point only a small fraction of A beta is in the co-aggregate. These weak transient interactions kinetically redirect the aggregation prone A beta from self-assembling into amyloid fibrils. These findings suggest that even such weak binders might be effective as therapeutics against pathogenic protein aggregation.
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| 4. |
- Abelein, Axel, et al.
(författare)
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Transient small molecule interactions kinetically modulate amyloid β peptide self-assembly.
- 2012
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Ingår i: FEBS Letters. - : Elsevier. - 0014-5793 .- 1873-3468. ; 586:22, s. 3991-3995
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Tidskriftsartikel (refereegranskat)abstract
- Small organic molecules, like Congo red and lacmoid, have been shown to modulate the self-assembly of the amyloid β peptide (Aβ). Here, we show that Aβ forms NMR invisible non-toxic co-aggregates together with lacmoid as well as Congo red. We find that the interaction involves two distinct kinetic processes and at every given time point only a small fraction of Aβ is in the co-aggregate. These weak transient interactions kinetically redirect the aggregation prone Aβ from self-assembling into amyloid fibrils. These findings suggest that even such weak binders might be effective as therapeutics against pathogenic protein aggregation.
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| 5. |
- Almandoz-Gil, Leire, et al.
(författare)
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Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways
- 2017
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Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849 .- 1873-4596. ; 110, s. 421-431
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Tidskriftsartikel (refereegranskat)abstract
- Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96 h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.
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| 6. |
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| 7. |
- Atapour, Masoud, et al.
(författare)
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Metal release from stainless steel 316L in whey protein - And simulated milk solutions under static and stirring conditions
- 2019
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Ingår i: Food Control. - : ELSEVIER SCI LTD. - 0956-7135 .- 1873-7129. ; 101, s. 163-172
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Tidskriftsartikel (refereegranskat)abstract
- Stainless steel is an important transport and processing contact material for bovine milk and dairy products. Release (migration) of metals, ions, complexes or wear debris/particles, and metal-induced protein aggregation in such environments are hence important to consider both from a corrosion and food safety perspective. This study aims on investigating the release of iron (Fe), chromium (Cr), and nickel (Ni) from AISI 316L stainless steel in contact with whey protein solutions relevant for protein drinks, and on how the whey proteins are influenced by stirring with a magnetic stir bar and metal release. Mechanistic insight is gained by parallel investigations of metal release from two reference non-protein containing solutions, a metal-complexing (citrate-containing) simulated milk solution (SMS) and a low complexing phosphate buffered saline solution (PBS). All immersion exposures were conducted at pH 6.8 for 0.5, 4, 24 and 48 hat room temperature at static and stirring conditions. All solutions and samples were investigated using different chemical, spectroscopic, microscopic, and electrochemical methods. Significantly higher amounts of Fe, Cr, and Ni were released into the whey protein solution (80 g/L) as compared to SMS and PBS. Strong enrichment of Cr in the surface oxide and reduction of the surface oxide thickness were associated with a higher amount of Ni release in the metal-complexing solutions (SMS and whey protein) compared with PBS. Stirring conditions resulted in higher amounts of metal release, enrichment of Cr in the surface oxide, and clear signs of wear of the 316L surface in all solutions compared to static conditions. The wear mechanism in the whey protein solution was different as compared to corresponding processes in SMS and PBS, involving an etching-like process and larger-sized wear debris. Electrochemical measurements at static conditions confirmed observed differences between the solutions, with the lowest corrosion resistance observed for coupons exposed in the whey protein solution, followed by SMS and PBS. Released metals in solution from the 316L coupons in contact with the whey protein solution resulted in enhanced rates of protein aggregation and precipitation of protein aggregates from solution. Further studies should be made to investigate other relevant test conditions and assess toxicological risks.
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| 8. |
- Bäcklund, Fredrik
(författare)
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Preparation and Application of Functionalized Protein Fibrils
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many proteins have an innate ability to self-assemble into fibrous structures known as amyloid fibrils. From a material science perspective, fibrils have several interesting characteristics, including a high stability, a distinct shape and tunable surface properties. Such structures can be given additional properties through functionalization by other compounds such as fluorophores. Combination of fibrils with a function yielding compound can be achieved in several ways. Covalent bond attachment is specific, but cumbersome. External surface adhesion is nonspecific, but simple. However, in addition, internal non-covalent functionalization is possible. In this thesis, particular emphasis is put on internal functionalization of fibrils; by co-grinding fibril forming proteins with a hydrophobic molecule, a protein-hydrophobic compound molecule composite can be created that retains the proteins innate ability to form fibrils. Subsequently formed fibrils will thus have the structural properties of the protein fibril as well as the properties of the incorporated compound. The functionalization procedures used throughout this thesis are applicable for a wide range of chromophores commonly used for organic electronics and photonics. The methods developed and the prepared materials are useful for applications within optoelectronics as well as biomedicine.Regardless of the methodology of functionalization, using functionalized fibrils in a controlled fashion for material design requires an intimate understanding of the formation process and knowledge of the tools available to control not only the formation but also any subsequent macroscale assembly of fibrils. The development and application of such tools are described in several of the papers included in this thesis. With the required knowledge in hand, the possible influence of fibrils on the functionalizing agents, and vice versa, can be probed. The characteristic traits of the functionalized fibril can be customized and the resulting material can be organized and steered towards a specific shape and form. This thesis describes how control over the process of formation, functionalization and organization of functionalized fibrils can be utilized to influence the hierarchical assembly of fibrils – ranging from spherical structures to spirals; the function – fluorescent or conducting; and macroscopic properties – optical birefringence and specific arrangement of functionalized fibrils in the solid state. In conclusion, the use of amyloid fibrils in material science has great potential. Herein is presented a possible route towards a fully bottom up approach ranging from the nanoscale to the macroscale.
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| 9. |
- Cai, Yixiao, et al.
(författare)
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Changes in secondary structure of alpha-synuclein during oligomerization induced by reactive aldehydes
- 2015
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 464:1, s. 336-341
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Tidskriftsartikel (refereegranskat)abstract
- The oxidative stress-related reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown to promote formation of alpha-synuclein oligomers in vitro. However, the changes in secondary structure of alpha-synuclein and the kinetics of the oligomerization process are not known and were the focus of this study. Size exclusion chromatography showed that after 1 h of incubation, HNE induced the formation of an oligomeric alpha-synuclein peak with a molecular weight of about similar to 2000 kDa, which coincided with a decreasing similar to 50 kDa monomeric peak. With prolonged incubation (up to 24 h) the oligomeric peak became the dominating molecular species. In contrast, in the presence of ONE, a similar to 2000 oligomeric peak was exclusively observed after 15 min of incubation and this peak remained constant with prolonged incubation. Western blot analysis of HNE-induced alpha-synuclein oligomers showed the presence of monomers (15 kDa), SDS-resistant low molecular (30-160 kDa) and high molecular weight oligomers (>= 260 kDa), indicating that the oligomers consisted of both covalent and non-covalent protein. In contrast, ONE-induced alpha-synuclein oligomers only migrated as covalent cross-linked high molecular-weight material (>= 300 kDa). Both circular dichroism (CD) and Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the formation of HNE- and ONE-induced oligomers coincided with a spectral change from random coil to beta-sheet. However, ONE-induced alpha-synuclein oligomers exhibited a slightly higher degree of beta-sheet. Taken together, our results indicate that both HNE and ONE induce a change from random coil to beta-sheet structure that coincides with the formation of alpha-synuclein oligomers; albeit through different kinetic pathways depending on the degree of cross-linking. (C) 2015 Elsevier Inc. All rights reserved.
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| 10. |
- Chaudhary, Himanshu, et al.
(författare)
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Dissecting the structural organization of multiprotein amyloid aggregates using a bottom-up approach
- 2020
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:10, s. 1447-1457
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Tidskriftsartikel (refereegranskat)abstract
- Deposition of fibrillar amyloid β (Aβ) in senile plaques is a pathological signature of Alzheimer's disease. However, senile plaques also contain many other components, including a range of different proteins. Although the composition of the plaques can be analyzed in post mortem tissue, knowledge of the molecular details of these multiprotein inclusions and their assembly processes is limited, which impedes the progress in deciphering the biochemical mechanisms associated with Aβ pathology. We here describe a bottom-up approach to monitor how proteins from human cerebrospinal fluid associate with Aβ amyloid fibrils to form plaque particles. The method combines flow cytometry and mass spectrometry proteomics and allowed us to identify and quantify 128 components of the captured multiprotein aggregates. The results provide insights in the functional characteristics of the sequestered proteins and reveal distinct interactome responses for the two investigated Aβ variants, Aβ(1-40) and Aβ(1-42). Furthermore, the quantitative data is used to build models of the structural organization of the multiprotein aggregates, which suggests that Aβ is not the primary binding target for all the proteins; secondary interactions account for the majority of the assembled components. The study elucidates how different proteins are recruited into senile plaques and establishes a new model system for exploring the pathological mechanisms of Alzheimer's disease from a molecular perspective.
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| 11. |
- Chaudhary, Himanshu, et al.
(författare)
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Intrinsically disordered protein as carbon nanotube dispersant: How dynamic interactions lead to excellent colloidal stability
- 2019
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Ingår i: Journal of Colloid and Interface Science. - : Academic Press. - 0021-9797 .- 1095-7103. ; 556, s. 172-179
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Tidskriftsartikel (refereegranskat)abstract
- The rich pool of protein conformations combined with the dimensions and properties of carbon nanotubes create new possibilities in functional materials and nanomedicine. Here, the intrinsically disordered protein α-synuclein is explored as a dispersant of single-walled carbon nanotubes (SWNTs) in water. We use a range of spectroscopic methods to quantify the amount of dispersed SWNT and to elucidate the binding mode of α-synuclein to SWNT. The dispersion ability of α-synuclein is good even with mild sonication and the obtained dispersion is very stable over time. The whole polypeptide chain is involved in the interaction accompanied by a fraction of the chain changing into a helical structure upon binding. Similar to other dispersants, we observe that only a small fraction (15–20%) of α-synuclein is adsorbed on the SWNT surface with an average residence time below 10 ms
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| 12. |
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| 13. |
- De Oliveira, Danilo Hirabae
(författare)
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Integrative structural biology of protein fibers: Spider silk and beta-lactoglobulin nanofibrils
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins found in nature offer a vast range of exceptional materials, including high-performancebiopolymers such as spider silks and whey protein nanofibrils. Fibrous proteins possess immensepotential for developing novel materials suited for various applications, such as medicalbiomaterials or industrial products. This thesis uses an interdisciplinary approach based onexperimental and computational methods to present insights into the fundamental aspects ofprotein fibers, exploring details on the molecular level and their self-assembly.Spider silk threads exhibit strength, elasticity, and the ability to withstand high-energy loads.Additionally, silk is naturally degradable, and compatible with cell growth, and non-immunogenic.This thesis examines the molecular assembly of spider silk, particularly the secondary structurelevel, which contributes greatly to its properties. We unveil the structural details of therecombinant spidroins 4RepCT and the CT domain over hydrophobic/hydrophilic surfaces,describing their periodic oriented macrostructure and stability. Furthermore, it is reported that theCT domain form β-nanocrystalline components, revealing a specific segment (helix No4) that canself-assemble into nanofibrils in a pH-sensitive manner. In addition, we describe the method ofsortase-mediated transpeptidation reaction used to catalyze the covalent coupling of the spidroins4Rep and CT, resulting in partially isotopically labeled fibers suitable for solid-state NMRspectroscopy analyses.β-lactoglobulin is an emerging protein source used to create advanced biomaterials because of itshigh availability and ability to assemble to protein nanofibrils (PNFs). Recombinant and syntheticβ-lactoglobulin PNFs with isotopic labelling are generated and analyzed using solid-state NMRspectroscopy and atomic force microscopy. The fibrils of both species present congruenciesregarding morphology with unbranched conformation and a height of approximately 6 nm. At thesame time, their NMR spectra demonstrate accordance with their hydrophobic residues (i.e., Ala,Val, Ile, and Leu) as β-sheets. In addition, distinct inter-residue cross-peaks of Ser-Thr and LeuIle provide insights into the molecular structure of β-lactoglobulin PNF.This thesis presents new knowledge about the hierarchy of protein fibrils and the structure ofprotein-based materials at the molecular level. This knowledge can unlock the design anddevelopment of innovative protein-based materials for various applications.
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| 14. |
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| 15. |
- De Oliveira, Danilo Hirabae, et al.
(författare)
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Structural conversion of the spidroin C-terminal domain during assembly of spider silk fibers
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into β-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.
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| 16. |
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| 17. |
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| 18. |
- Dincbas-Renqvist, Vildan, et al.
(författare)
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Thermodynamics of folding, stabilization, and binding in an engineered protein--protein complex.
- 2004
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 126:36, s. 11220-30
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Tidskriftsartikel (refereegranskat)abstract
- We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA)(-)(1) affibody and it's Z domain binding partner as a model. Free Z(SPA)(-)(1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA)(-)(1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA)(-)(1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA)(-)(1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA)(-)(1) complex is associated with a large change in conformational entropy that opposes binding.
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| 19. |
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| 20. |
- Dogan, Jakob, et al.
(författare)
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Thermodynamics of folding and binding in an affibody:affibody complex.
- 2006
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 359:5, s. 1305-15
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Tidskriftsartikel (refereegranskat)abstract
- Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K(d)=0.1 microM. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T(M) approximately 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with DeltaH degrees (bind) = -9.0 (+/-0.1) kcal mol(-1)(.) The heat capacity change DeltaC(P) degrees (,bind)=-0.43 (+/-0.01) kcal mol(-1) K(-1) is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-TDeltaS degrees (bind) = -0.6 (+/-0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.
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| 21. |
- Dubnovitsky, Anatoly, et al.
(författare)
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Amyloid-beta Protofibrils: Size, Morphology and Synaptotoxicity of an Engineered Mimic
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Structural and biochemical studies of the aggregation of the amyloid-beta peptide (A beta) are important to understand the mechanisms of Alzheimer's disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of A beta called A beta cc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of A beta(42)cc protofibrils. Like wild type A beta they appear as smooth rod-like particles with a diameter of 3.1 (+/- 0.2) nm and typical lengths in the range 60 to 220 nm when observed by atomic force microscopy. Non-perturbing analytical ultracentrifugation and nanoparticle tracking analyses are consistent with such rod-like protofibrils. A beta(42)cc protofibrils bind the ANS dye indicating that they, like other toxic protein aggregates, expose hydrophobic surface. Assays with the OC/A11 pair of oligomer specific antibodies put A beta(42)cc protofibrils into the same class of species as fibrillar oligomers of wild type A beta. A beta(42)cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, A beta(42)cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by A beta(42)cc and aggregates of wild type A beta(42). We suggest that A beta(42)cc protofibrils may be used in research and applications that require stable preparations of protofibrillar A beta.
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| 22. |
- Ekblad, Torun, et al.
(författare)
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Synthesis and chemoselective intramolecular cross-linking of a HER2-binding Affibody
- 2009
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 92:2, s. 116-123
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Tidskriftsartikel (refereegranskat)abstract
- The human epidermal growth factor receptor HER2 has emerged as an important target for molecular imaging of breast cancer. This article presents the design and synthesis of a HER2-targeting affibody molecule with improved stability and tumor targeting capacity, and with potential use as an imaging agent. The 58 aa three-helix bundle protein was assembled using solid-phase peptide synthesis, and a chemoselective ligation strategy was used to establish an intramolecular thioether bond between the side chain thiol group of a cysteine residue, positioned in the loop between helices I and II, and a chloroacetyl group on the side chain amino group of the C-terminal lysine residue. The tethered protein offered an increased thermal stability, with a melting temperature of 64 degrees C, compared to 54 degrees C for the linear control. The ligation did not have a major influence on the HER2 binding affinity, which was 320 and 380 pM for the crosslinked and linear molecules, respectively. Biodistribution studies were performed both in normal and tumor-bearing mice to evaluate the impact of the crosslinking on the in vivo behavior and on the tumor targeting performance. The distribution pattern was characterized by a low uptake in all organs except kidney, and rapid clearance from blood and normal tissue. Crosslinking of the protein resulted in a significantly increased tumor accumulation, rendering the tethered HER2-binding affibody molecule a valuable lead in the development of superior HER2 imaging agents.
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| 23. |
- Feldwisch, Joachim, et al.
(författare)
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Design of an optimized scaffold for affibody molecules.
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 398:2, s. 232-247
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K(D), of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.
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| 24. |
- Gowda, Vasantha, et al.
(författare)
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Structural characterisation of amyloid-like fibrils formed by an amyloidogenic peptide segment of β-lactoglobulin
- 2021
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Ingår i: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 11:45, s. 27868-27879
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Tidskriftsartikel (refereegranskat)abstract
- Protein nanofibrils (PNFs) represent a promising class of biobased nanomaterials for biomedical and materials science applications. In the design of such materials, a fundamental understanding of the structure–function relationship at both molecular and nanoscale levels is essential. Here we report investigations of the nanoscale morphology and molecular arrangement of amyloid-like PNFs of a synthetic peptide fragment consisting of residues 11–20 of the protein β-lactoglobulin (β-LG11–20), an important model system for PNF materials. Nanoscale fibril morphology was analysed by atomic force microscopy (AFM) that indicates the presence of polymorphic self-assembly of protofilaments. However, observation of a single set of 13C and 15N resonances in the solid-state NMR spectra for the β-LG11–20 fibrils suggests that the observed polymorphism originates from the assembly of protofilaments at the nanoscale but not from the molecular structure. The secondary structure and inter-residue proximities in the β-LG11–20 fibrils were probed using NMR experiments of the peptide with 13C- and 15N-labelled amino acid residues at selected positions. We can conclude that the peptides form parallel β-sheets, but the NMR data was inconclusive regarding inter-sheet packing. Molecular dynamics simulations confirm the stability of parallel β-sheets and suggest two preferred modes of packing. Comparison of molecular dynamics models with NMR data and calculated chemical shifts indicates that both packing models are possible.
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| 25. |
- Gräslund, Torbjörn, et al.
(författare)
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IGF-1R binding polypeptides and their use
- 2007
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Patent (populärvet., debatt m.m.)abstract
- This invention relates to polypeptides which bind to IGF-1R and to applications of those polypeptides in medicine, veterinary medicine, diagnostics and imaging.
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| 26. |
- Hedberg, Yolanda, Docent, 1985-, et al.
(författare)
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Synergistic effects of metal-induced aggregation of human serum albumin
- 2019
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Ingår i: Colloids and Surfaces B. - : ELSEVIER SCIENCE BV. - 0927-7765 .- 1873-4367. ; 173, s. 751-758
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Tidskriftsartikel (refereegranskat)abstract
- Exposure to cobalt (Co), chromium (Cr), and nickel (Ni) occurs often via skin contact and from different dental and orthopedic implants. The metal ions bind to proteins, which may induce structural changes and aggregation, with different medical consequences. We investigated human serum albumin (HSA) aggregation in the presence of Co-II, Cr-III, and/or Ni-II ions and/or their nanoparticle precipitates by using scattering, spectroscopic, and imaging techniques, at simulated physiological conditions (phosphate buffered saline - PBS, pH 7.3) using metal salts that did not affect the pH, and at HSA:metal molar ratios of up to 1:8. Co ions formed some solid nano particles in PBS at the investigated conditions, as determined by nanoparticle tracking analysis, but the Cr-III anions and Ni-II ions remained fully soluble. It was found that all metal ions induced HSA aggregation, and this effect was significantly enhanced when a mixture of all three metal ions was present instead of any single type of ion. Thus, the metal ions induce aggregation synergistically. HSA aggregates formed linear structures on a mica surface in the presence of Cr-III ions. A clear tendency of aggregation and linearly aligned aggregates was seen in the presence of all three metal ions. Spectroscopic investigations indicated that the majority of the HSA molecules maintained their alpha helical secondary structure and conformation. This study highlights the importance of synergistic effects of metal ions and/or their precipitates on protein aggregation, which are highly relevant for implant materials and common exposures to metals.
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| 27. |
- Herneke, A., et al.
(författare)
-
Protein Nanofibrils for Sustainable Food-Characterization and Comparison of Fibrils from a Broad Range of Plant Protein Isolates
- 2021
-
Ingår i: ACS Food Science and Technology. - : American Chemical Society (ACS). - 2692-1944. ; 1:5, s. 854-864
-
Tidskriftsartikel (refereegranskat)abstract
- Protein nanofibrils (PNFs) from plant-based protein sources have great potential for use in new sustainable food applications or biobased materials. Plant-based proteins from seven different sources (fava bean, mung bean, lupin, oat, rapeseed, soybean, and potato) were evaluated here for their ability for forming PNFs and compared with whey protein PNFs. Formation of PNFs was studied under incubation at acidic conditions (pH 2) and heat (85-90 °C) for 24-96 h. Presence of PNFs was detected using thioflavin T, circular dichroism spectroscopy, Fourier-transform infrared spectroscopy, and atomic force microscopy. The results showed that all plant-based proteins were able to form PNFs. For some of the plant protein isolates in this study, nanofibril formation is reported for the first time. We also describe and compare the distinct features associated with each protein source and the morphological alterations of the nanoscale structures. Finally, we describe how PNF production can be enhanced by purifying the fibril-forming protein component. Taken together, our study suggests that the structural and functional variation within plant protein nanofibrils can be exploited in future scaled-up food or material applications.
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| 28. |
- Herneke, Anja, et al.
(författare)
-
Protein Nanofibrils from Fava Bean and Its Major Storage Proteins : Formation and Ability to Generate and Stabilise Foams
- 2023
-
Ingår i: Foods. - : MDPI AG. - 2304-8158. ; 12:3
-
Tidskriftsartikel (refereegranskat)abstract
- Protein nanofibrils (PNFs) have potential for use in food applications as texture inducers. This study investigated the formation of PNFs from protein extracted from whole fava bean and from its two major storage proteins, globulin fractions 11S and 7S. PNFs were formed by heating (85 degrees C) the proteins under acidic conditions (pH 2) for 24 h. Thioflavin T fluorescence and atomic force microscopy techniques were used to investigate PNF formation. The foaming properties (capacity, stability, and half-life) were explored for non-fibrillated and fibrillated protein from fava bean, 11S, and 7S to investigate the texturing ability of PNFs at concentrations of 1 and 10 mg/mL and pH 7. The results showed that all three heat-incubated proteins (fava bean, 11S, and 7S) formed straight semi-flexible PNFs. Some differences in the capacity to form PNFs were observed between the two globulin fractions, with the smaller 7S protein being superior to 11S. The fibrillated protein from fava bean, 11S, and 7S generated more voluminous and more stable foams at 10 mg/mL than the corresponding non-fibrillated protein. However, this ability for fibrillated proteins to improve the foam properties seemed to be concentration-dependent, as at 1 mg/mL, the foams were less stable than those made from the non-fibrillated protein.
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| 29. |
- Herneke, Anja, et al.
(författare)
-
Protein nanofibrils from mung bean : The effect of pH on morphology and the ability to form and stabilise foams
- 2023
-
Ingår i: Food Hydrocolloids. - : Elsevier BV. - 0268-005X .- 1873-7137. ; 136
-
Tidskriftsartikel (refereegranskat)abstract
- Protein nanofibrils (PNFs) have potential food uses due to their favorable mechanical and rheological properties. In order to use plant-based protein nanofibrils (PNFs) in new sustainable food applications, a better under-standing is needed of the impact of pH on PNFs and their functional properties. In this study, we developed an improved method for generating PNFs from mung bean protein isolate and its globular 8S fraction. We then investigated how these PNFs are affected by increased pH and how pH affects their ability to form stable foams. PNFs were generated in acidic conditions (pH 2) by heating at 85 degrees C for 2-48 h. Formation of PNFs and the effect of increased pH on their stability were evaluated using thioflavin T fluorescence, electrophoretic gel separation, circular dichroism spectroscopy, atomic force microscopy and viscosity profiling. Foams were made by intense mixing with a homogeniser and evaluated for foam capacity, foam stability and bubble size distribution, using confocal imaging. The results showed that it is possible to optimise the fibrillation conditions for mung bean by generating a more pure initial protein isolate by salt extraction. The results also showed that pH alters the structure of PNFs by degradation and aggregation around the isoelectric point of the protein isolate. At neutral pH, the PNFs were slightly shorter than at the starting pH, but no longer formed aggregates. Fibrillated mung bean protein at pH 4-8 was found to have good foaming properties compared with non-fibrillated protein at the same conditions. The new knowledge generated in this study about how pH alters the structure and foaming properties of PNFs can pave the way for use of PNFs in new innovative food applications.
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| 30. |
- Hober, Sophia, Professor, 1965-, et al.
(författare)
-
Systematic evaluation of SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay
- 2021
-
Ingår i: Clinical & Translational Immunology. - : Wiley. - 2050-0068. ; 10:7
-
Tidskriftsartikel (refereegranskat)abstract
- Objective. The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. Methods. More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. Results. Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. Conclusion. These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.
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| 31. |
- Härd, Torleif, et al.
(författare)
-
Inhibition of Amyloid Formation
- 2012
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 421, s. 441-465
-
Tidskriftsartikel (refereegranskat)abstract
- Amyloid is aggregated protein in the form of insoluble fibrils. Amyloid deposition in human tissue-amyloidosis-is associated with a number of diseases including all common dementias and type II diabetes. Considerable progress has been made to understand the mechanisms leading to amyloid formation. It is, however, not yet clear by which mechanisms amyloid and protein aggregates formed on the path to amyloid are cytotoxic. Strategies to prevent protein aggregation and amyloid formation are nevertheless, in many cases, promising and even successful. This review covers research on intervention of amyloidosis and highlights several examples of how inhibition of protein aggregation and amyloid formation has been achieved in practice. For instance, rational design can provide drugs that stabilize a native folded state of a protein, protein engineering can provide new binding proteins that sequester monomeric peptides from aggregation, small molecules and peptides can be designed to block aggregation or direct it into non-cytotoxic paths, and monoclonal antibodies have been developed for therapies towards neurodegenerative diseases based on inhibition of amyloid formation and clearance. (c) 2012 Elsevier Ltd. All rights reserved.
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| 32. |
- Josefsson, Leila, et al.
(författare)
-
Potato Protein Nanofibrils Produced from a Starch Industry Sidestream
- 2020
-
Ingår i: ACS Sustainable Chemistry and Engineering. - : AMER CHEMICAL SOC. - 2168-0485. ; 8:2, s. 1058-1067
-
Tidskriftsartikel (refereegranskat)abstract
- Protein nanofibrils have emerged as promising building blocks in functional bio/nanomaterials as well as in food products. We here demonstrate that nanofibrils with amyloid-like properties can be produced from potato protein isolate, a major sidestream from the starch industry. Methods for solubilization of potato proteins are evaluated, and a protocol for the assembly of protein nanofibrils is presented. Characterization of the nanofibrils shows that they are rich in beta-sheet structure and display the cross-beta X-ray fiber diffraction pattern, which is a hallmark of amyloid-like fibrils. Atomic force microscopy shows that the fibrils are ca. 4-5 nm in diameter with a nanoscale morphology that displays a high degree of curvature. Using mass spectrometry we identify four peptides that constitute the core building blocks of the nanofibrils and show that they originate from two different classes of proteins. The structural characteristics of these peptides are distinct from previously studied plant protein nanofibrils and thereby reveal new knowledge about the formation of protein nanostructures from agricultural resources.
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| 33. |
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| 34. |
- Josefsson, Leila, et al.
(författare)
-
Structural basis for the formation of soy protein nanofibrils
- 2019
-
Ingår i: RSC Advances. - : ROYAL SOC CHEMISTRY. - 2046-2069. ; 9:11, s. 6310-6319
-
Tidskriftsartikel (refereegranskat)abstract
- Amyloid-like protein nanofibrils (PNFs) can assemble from a range of different proteins including disease-associated proteins, functional amyloid proteins and several proteins for which the PNFs are neither related to disease nor function. We here examined the core building blocks of PNFs formed by soy proteins. Fibril formation at pH 2 and 90 degrees C is coupled to peptide hydrolysis which allows isolation of the PNF-forming peptides and identification of them by mass spectrometry. We found five peptides that constitute the main building blocks in soy PNFs, three of them from the protein b-conglycinin and two from the protein glycinin. The abilities of these peptides to form PNFs were addressed by amyloid prediction software and by PNF formation of the corresponding synthetic peptides. Analysis of the structural context in the native soy proteins revealed two structural motifs for the PNF-forming peptides: (i) so-called b-arches and (ii) helical segments involved in quaternary structure contacts. However, the results suggest that neither the native structural motifs nor the protein of origin defines the morphology of the PNFs formed from soy protein isolate.
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| 35. |
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| 36. |
- Kamada, Ayaka, et al.
(författare)
-
Flow-assisted assembly of nanostructured protein microfibers
- 2017
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : PNAS. - 0027-8424 .- 1091-6490. ; 114:6, s. 1232-1237
-
Tidskriftsartikel (refereegranskat)abstract
- Some of the most remarkable materials in nature are made from proteins. The properties of these materials are closely connected to the hierarchical assembly of the protein building blocks. In this perspective, amyloid-like protein nanofibrils (PNFs) have emerged as a promising foundation for the synthesis of novel bio-based materials for a variety of applications. Whereas recent advances have revealed the molecular structure of PNFs, the mechanisms associated with fibril-fibril interactions and their assembly into macroscale structures remain largely unexplored. Here, we show that whey PNFs can be assembled into microfibers using a flow-focusing approach and without the addition of plasticizers or cross-linkers. Microfocus small-angle X-ray scattering allows us to monitor the fibril orientation in the microchannel and compare the assembly processes of PNFs of distinct morphologies. We find that the strongest fiber is obtained with a sufficient balance between ordered nanostructure and fibril entanglement. The results provide insights in the behavior of protein nanostructures under laminar flow conditions and their assembly mechanism into hierarchical macroscopic structures.
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| 37. |
- Kamada, Ayaka, et al.
(författare)
-
Hierarchical propagation of structural features in protein nanomaterials
- 2022
-
Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 14:6, s. 2502-2510
-
Tidskriftsartikel (refereegranskat)abstract
- Natural high-performance materials have inspired the exploration of novel materials from protein building blocks. The ability of proteins to self-organize into amyloid-like nanofibrils has opened an avenue to new materials by hierarchical assembly processes. As the mechanisms by which proteins form nanofibrils are becoming clear, the challenge now is to understand how the nanofibrils can be designed to form larger structures with defined order. We here report the spontaneous and reproducible formation of ordered microstructure in solution cast films from whey protein nanofibrils. The structural features are directly connected to the nanostructure of the protein fibrils, which is itself determined by the molecular structure of the building blocks. Hence, a hierarchical assembly process ranging over more than six orders of magnitude in size is described. The fibril length distribution is found to be the main determinant of the microstructure and the assembly process originates in restricted capillary flow induced by the solvent evaporation. We demonstrate that the structural features can be switched on and off by controlling the length distribution or the evaporation rate without losing the functional properties of the protein nanofibrils.
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| 38. |
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| 39. |
- Lendel, Christofer, et al.
(författare)
-
3D J-resolved NMR spectroscopy for unstructured polypeptides : fast measurement of 3J HNH alpha coupling constants with outstanding spectral resolution.
- 2009
-
Ingår i: Journal of biomolecular NMR. - : Springer Science and Business Media LLC. - 1573-5001 .- 0925-2738. ; 44:1, s. 35-42
-
Tidskriftsartikel (refereegranskat)abstract
- A powerful experiment for the investigation of conformational properties of unstructured states of proteins is presented. The method combines a phase sensitive J-resolved experiment with a (1)H-(15)N SOFAST-HMQC to provide a 3D spectrum with an E.COSY pattern originating from splittings due to (3)J(HNH alpha) and (2)J(NH alpha) couplings. Thereby an effectively homodecoupled (1)H-(15)N correlation spectrum is obtained with significantly improved resolution and greatly reduced spectral overlap compared to standard HSQC and HMQC experiments. The (3)J(HNH alpha) is revealed in three independent ways directly from the peak positions, allowing for internal consistency testing. In addition, the natural H(N) linewidths can easily be extracted from the lineshapes. Thanks to the SOFAST principle, the limited sweep width needed in the J-dimension and the short phase cycle, data accumulation is rapid with excellent sensitivity per time unit. The experiment is demonstrated for the intrinsically unstructured 14 kDa protein alpha-synuclein.
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| 40. |
- Lendel, Christofer, et al.
(författare)
-
A Hexameric Peptide Barrel as Building Block of Amyloid-β Protofibrils
- 2014
-
Ingår i: Angewandte Chemie. - : Wiley. - 0044-8249 .- 1521-3757. ; 126:47, s. 12970-12974
-
Tidskriftsartikel (refereegranskat)abstract
- Oligomeric and protofibrillar aggregates formed by the amyloid-β peptide (Aβ) are believed to be involved in the pathology of Alzheimer’s disease. Central to Alzheimer pathology is also the fact that the longer Aβ42 peptide is more prone to aggregation than the more prevalent Aβ40. Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ42 forms oligomers and protofibrils more easily than Aβ40.
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| 41. |
- Lendel, Christofer, 1976-, et al.
(författare)
-
A Hexameric Peptide Barrel as Building Block of Amyloid-β Protofibrils
- 2014
-
Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 53:47, s. 12756-12760
-
Tidskriftsartikel (refereegranskat)abstract
- Oligomeric and protofibrillar aggregates formed by the amyloid-β peptide (Aβ) are believed to be involved in the pathology of Alzheimer’s disease. Central to Alzheimer pathology is also the fact that the longer Aβ42 peptide is more prone to aggregation than the more prevalent Aβ40. Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ42 forms oligomers and protofibrils more easily than Aβ40.
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| 42. |
- Lendel, Christofer, et al.
(författare)
-
Biophysical characterization of Z(SPA-1)--a phage-display selected binder to protein A.
- 2004
-
Ingår i: Protein science : a publication of the Protein Society. - : Wiley. - 0961-8368 .- 1469-896X. ; 13:8, s. 2078-88
-
Tidskriftsartikel (refereegranskat)abstract
- Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.
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| 43. |
- Lendel, Christofer, et al.
(författare)
-
Combined Solution- and Magic Angle Spinning NMR Reveals Regions of Distinct Dynamics in Amyloid beta Protofibrils
- 2016
-
Ingår i: Chemistryselect. - : Wiley. - 2365-6549. ; 1:18, s. 5850-5853
-
Tidskriftsartikel (refereegranskat)abstract
- Solid-state magic angle spinning (MAS) NMR has emerged as an important tool for investigations of protein aggregates and amyloid fibrils, which are not accessible for solution NMR experiments. We recently presented a structural model for amyloid beta (A beta) protofibrils based on MAS-NMR data. The absence of resonances for the N-terminus of A beta in this dataset suggested that it might be disordered and more dynamic than the structural core. We here provide evidence for a distinct dynamic regime in the N-terminal part of the peptide and show that the structural characteristics of this region can be elucidated using C-13-detected solution NMR. The results shed more light on the structural properties of pre-fibrillar A beta species and demonstrate the potential of combining MAS and solution NMR experiments for the characterization of structure and dynamics of complex protein assemblies.
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| 44. |
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| 45. |
- Lendel, Christofer, et al.
(författare)
-
Detergent-like interaction of Congo red with the amyloid beta peptide
- 2010
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:7, s. 1358-1360
-
Tidskriftsartikel (refereegranskat)abstract
- Accumulating evidence links prefibrillar oligomeric species of the amyloid beta peptide (Abeta) to cellular toxicity in Alzheimer's disease, potentially via disruption of biological membranes. Congo red (CR) affects protein aggregation. It is known to self-associate into micelle-like assemblies but still reduces the toxicity of Abeta aggregates in cell cultures and model organisms. We show here that CR interacts with Abeta(1-40) in a manner similar to that of anionic detergents. Although CR promotes beta sheet formation and peptide aggregation, it may also solubilize toxic protein species, making them less harmful to critical cellular components and thereby reducing amyloid toxicity.
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| 46. |
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| 47. |
- Lendel, Christofer
(författare)
-
Molecular principles of protein stability and protein-protein interactions
- 2005
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free ZSPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free ZSPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general.
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| 48. |
- Lendel, Christofer, et al.
(författare)
-
Protein nanofibrils and their use as building blocks of sustainable materials
- 2021
-
Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 11:62, s. 39188-39215
-
Forskningsöversikt (refereegranskat)abstract
- The development towards a sustainable society requires a radical change of many of the materials we currently use. Besides the replacement of plastics, derived from petrochemical sources, with renewable alternatives, we will also need functional materials for applications in areas ranging from green energy and environmental remediation to smart foods. Proteins could, with their intriguing ability of self-assembly into various forms, play important roles in all these fields. To achieve that, the code for how to assemble hierarchically ordered structures similar to the protein materials found in nature must be cracked. During the last decade it has been demonstrated that amyloid-like protein nanofibrils (PNFs) could be a steppingstone for this task. PNFs are formed by self-assembly in water from a range of proteins, including plant resources and industrial side streams. The nanofibrils display distinct functional features and can be further assembled into larger structures. PNFs thus provide a framework for creating ordered, functional structures from the atomic level up to the macroscale. This review address how industrial scale protein resources could be transformed into PNFs and further assembled into materials with specific mechanical and functional properties. We describe what is required from a protein to form PNFs and how the structural properties at different length scales determine the material properties. We also discuss potential chemical routes to modify the properties of the fibrils and to assemble them into macroscopic structures.
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| 49. |
- Lendel, Christofer, et al.
(författare)
-
Structural basis for molecular recognition in an affibody:affibody complex.
- 2006
-
Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 359:5, s. 1293-304
-
Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K(d) approximately 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large ( approximately 1670 A(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine Halpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium N(eta)H(2) groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.
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| 50. |
- Liebau, Jobst, 1985-
(författare)
-
Membrane interactions of glycosyltransferases
- 2015
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many important biological processes occur near or in membranes. The role of membranes is not merely confined to compartmentalization, they also form the matrix for membrane associated proteins and are of functional importance. Membrane associated proteins on the other hand require specific membrane properties for proper function. The interactions between membranes and proteins are thus of paramount importance and are at the focus of this work.To draw valid conclusions about the nature of such interactions the membrane mimetics required in biophysical methods must faithfully mimic crucial properties of biological membranes. To this end, new types of small isotropic bicelles which mimic plant and bacterial membranes were characterized by their size and lipid dynamics using solution-state NMR. Small isotropic bicelles are specifically well suited for solution-state NMR studies since they maintain a bilayer while being sufficiently small to conduct interpretable experiments at the same time. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol, which are highly abundant in thylakoid membranes, were successfully incorporated into bicelles. Also, it was possible to make bicelles containing a lipid mixture extracted from Escherichia coli cells.A fundamental physical property of lipids in bilayers is their phase behaviour and thus the dynamics that lipids undergo in a membrane. Here, the dynamics of 13C-1H bonds in lipids were studied by nuclear spin relaxation. From such studies it was found that the glycerol backbone of lipids in bicelles is rigid while the flexibility of the acyl chain increases towards its end. Bulky head groups are rigid, while smaller head groups are more dynamic than the glycerol backbone. Acyl chain modifications, like unsaturations or cyclopropane moities, that are typically found in E. coli lipids, locally increase the rigidity of the acyl chain.Membrane interactions of a putative membrane anchor of the glycosyltransferase WaaG, MIR-WaaG, were studied by fluorescence methods, circular dichroism and solution-state NMR. It was found that MIR-WaaG binds to vesicles that mimic the anionic charge of E. coli inner membranes and that α-helical structure is induced upon interaction. The NMR-structure of MIR-WaaG agrees well with the crystal structure and from paramagnetic relaxation enhancement studies it could be concluded that a central part of MIR-WaaG is immersed in the membrane mimetic. Based on these results a model of the membrane interaction of WaaG is proposed where MIR-WaaG anchors WaaG to the cytosolic leaflet of the E. coli inner membrane via electrostatic interactions. These are potentially enhanced by membrane interactions of Tyr residues at the membrane interface and of hydrophobic residues inside the membrane.
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