| 1. |
- Härd, Torleif, et al.
(författare)
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Inhibition of Amyloid Formation
- 2012
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 421, s. 441-465
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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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| 2. |
- Crennell, SJ, et al.
(författare)
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Dimerisation and an increase in active site aromatic groups as adaptations to high temperatures: X-ray solution scattering and substrate-bound crystal structures of Rhodothermus marinus endoglucanase Cel12A
- 2006
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 356:1, s. 57-71
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Tidskriftsartikel (refereegranskat)abstract
- Cellulose, a polysaccharide consisting of beta-1,4-linked glucose, is the major component of plant cell walls and consequently one of the most abundant biopolymers on earth. Carbohydrate polymers such as cellulose are molecules with vast diversity in structure and function, and a multiplicity of hydrolases operating in concert are required for depolymerisation. The bacterium Rhodothermus marinus, isolated from shallow water marine hot springs, produces a number of carbohydrate-degrading enzymes including a family 12 cellulase Cel12A. The structure of R. marinus Cel12A in the ligand-free form (at 1.54 angstrom) and structures of RmCel12A after crystals were soaked in cellopentaose for two different lengths of time, have been determined. The shorter soaked complex revealed the conformation of unhydrolysed cellotetraose, while cellopentaose had been degraded more completely during the longer soak. Comparison of these structures with those of mesophilic family 12 cellulases in complex with inhibitors and substrate revealed that RmCel12A has a more extensive aromatic network in the active site cleft which ejects products after hydrolysis. The substrate structure confirms that during hydrolysis by family 12 cellulases glucose does not pass through a 2,5 B conformation. Small-angle X-ray scattering analysis of RmCel12A showed that the enzyme forms a loosely associated antiparallel dimer in solution, which may target the enzyme to the antiparallel polymer strands in cellulose. (c) 2005 Elsevier Ltd. All rights reserved.
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| 3. |
- Friedman, Mikaela, et al.
(författare)
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Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 376:5, s. 1388-1402
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Tidskriftsartikel (refereegranskat)abstract
- The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K(d)=5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K(d), was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K(d)=2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.
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| 4. |
- Holmner, Åsa, et al.
(författare)
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Blood group antigen recognition by Escherichia coli heat-labile enterotoxin
- 2007
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Ingår i: Journal of Molecular Biology. - London : Elsevier BV. - 0022-2836 .- 1089-8638. ; 371:3, s. 754-764
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Tidskriftsartikel (refereegranskat)abstract
- In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 angstrom crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAc alpha 3(Fuc alpha-2)Gal beta 4(Fuc alpha-3)Glc beta provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed. (c) 2007 Elsevier Ltd. All rights reserved.
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| 5. |
- Pozzo, Tania, et al.
(författare)
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Structural and Functional Analyses of beta-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3.
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 397, s. 724-739
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Tidskriftsartikel (refereegranskat)abstract
- Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized-that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 A resolution. Bgl3B from Thermot. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (alpha/beta)(8) domain similar to a triose phosphate isomerase barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second beta-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the -1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the -1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.
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| 6. |
- 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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| 7. |
- Kanje, Sara, 1986-, et al.
(författare)
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Protein engineering allows for mild affinity-based elution of therapeutic antibodies
- 2018
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:18, s. 3427-3438
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Tidskriftsartikel (refereegranskat)abstract
- Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification
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| 8. |
- Koos, Björn, et al.
(författare)
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Next-Generation Pathology : Surveillance of Tumor Microecology
- 2015
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:11, s. 2013-2022
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Forskningsöversikt (refereegranskat)abstract
- A tumor is a heterogeneous population of cells that provides an environment in which every cell resides in a microenvironmental niche. Microscopic evaluation of tissue sections, based on histology and immunohistochemistry, has been a cornerstone in pathology for decades. However, the dawn of novel technologies to investigate genetic aberrations is currently adopted in routine molecular pathology. We herein describe our view on how recent developments in molecular technologies, focusing on proximity ligation assay and padlock probes, can be applied to merge the two branches of pathology, allowing molecular profiling under histologic observation. We also discuss how the use of image analysis will be pivotal to obtain information at a cellular level and to interpret holistic images of tissue sections. By understanding the cellular communications in the microecology of tumors, we will be at a better position to predict disease progression and response to therapy.
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| 9. |
- Kurtovic, Sanela, et al.
(författare)
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Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 375:5, s. 1365-1379
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Tidskriftsartikel (refereegranskat)abstract
- A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.
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| 10. |
- Lendel, Christofer, et al.
(författare)
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Structural basis for molecular recognition 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. 1293-304
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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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