Should therefore, according to the proposed model, form soluble oligomers and protofibrils, but be unable to adopt the cross-b structure present in mature fibrils. We found that this indeed is the case: AbCC can readily form oligomeric species, which do not convert into fibrils unless the intramolecular disulfide bond is broken by reduction [16]. Protofibrils of Ab42CC are stable towards both dissociation (upon dilution) and fibril formation. More importantly, protofibrils formed by Ab42CC potentially constitute a tool for experiments in which stable protofibrils are required, such as for instance structural studies or immunization trials. Initial studies indicated a number of similarities betweenEngineered Ab42CC Protofibrils Mimic Wild Type Abwild type and Ab42CC protofibrils [16]. Here we characterize Ab42CC protofibrils in more detail, and compare them to wild type protofibrils, with regard to their morphology and size, surface properties and exposed antibody epitopes, protein binding, and their effect on synaptic activity in neurons.Materials and Methods Protein production and preparation of aggregatesAb40, Ab42 and Ab42CC were produced in E. coli by coexpression with the ZAb3 Ab-binding AffibodyH molecule as described previously [16,19]. The peptides were separated from the Affibody binder by denaturation in 7 M guanidinium chloride followed by immobilized metal affinity chromatography (IMAC) under denaturing conditions. Ab42CC protofibrils were obtained by separating oligomers with size exclusion chromatography (SEC) under native purchase CB 5083 buffer conditions [16], followed by concentration on a Vivaspin column (GE Healthcare) and heat treatment at 60uC for 10 min. Alternatively, protofibrils also form when guanidinium chloride is removed by dialysis of denatured Ab42CC at room temperature against 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl and 5 mM EDTA and a second dialysis for 4 to 6 hours in the same buffer without EDTA. Protofibrils of wild type Ab42 were identified in atomic force microscopy (AFM) images of an Ab42 aggregation reaction mixture. Ab42 monomer (,100 mM) in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl was kept at room temperature without shaking. A mixture of Ab42 protofibrils and smaller oligomers could be observed and distinguished in AFM images recorded after one day of incubation. The same solution was put in 37uC and shaking conditions for one more day in order to produce Ab42 fibrils for AFM imaging. Fibrils of wild type Ab40 and Ab42 for AFM and OC serum dot blot assays were prepared from similar mixtures that were subjected to 37uC and shaking to favor the formation of fibrils.equation solutions with a common (MedChemExpress 94-09-7 unimodal) frictional ratio. The partial specific volume of Ab42CC, and the buffer density and viscosity at 20uC, were calculated using the SEDNTERP program v 1.09 (Biomolecular Interaction Technologies Center, University of New Hampshire, Durham, NH; http://bitcwiki.sr.unh.edu/ index.php/Main_Page). Measured sedimentation coefficients were corrected to s20, w values (sedimentation coefficients in water at 20uC).Nanoparticle tracking analysis (NTA)Measurements were 23977191 performed with a NanoSight LM10-HS instrument (NanoSight, Amesbury, UK) equipped with an LM14 viewing unit using a 405 nm laser. Protofibrils of Ab42CC were diluted in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl to a final monomer concentration of 2 mM, and measurements were performed at 20uC for 30 s. The NanoSigh.Should therefore, according to the proposed model, form soluble oligomers and protofibrils, but be unable to adopt the cross-b structure present in mature fibrils. We found that this indeed is the case: AbCC can readily form oligomeric species, which do not convert into fibrils unless the intramolecular disulfide bond is broken by reduction [16]. Protofibrils of Ab42CC are stable towards both dissociation (upon dilution) and fibril formation. More importantly, protofibrils formed by Ab42CC potentially constitute a tool for experiments in which stable protofibrils are required, such as for instance structural studies or immunization trials. Initial studies indicated a number of similarities betweenEngineered Ab42CC Protofibrils Mimic Wild Type Abwild type and Ab42CC protofibrils [16]. Here we characterize Ab42CC protofibrils in more detail, and compare them to wild type protofibrils, with regard to their morphology and size, surface properties and exposed antibody epitopes, protein binding, and their effect on synaptic activity in neurons.Materials and Methods Protein production and preparation of aggregatesAb40, Ab42 and Ab42CC were produced in E. coli by coexpression with the ZAb3 Ab-binding AffibodyH molecule as described previously [16,19]. The peptides were separated from the Affibody binder by denaturation in 7 M guanidinium chloride followed by immobilized metal affinity chromatography (IMAC) under denaturing conditions. Ab42CC protofibrils were obtained by separating oligomers with size exclusion chromatography (SEC) under native buffer conditions [16], followed by concentration on a Vivaspin column (GE Healthcare) and heat treatment at 60uC for 10 min. Alternatively, protofibrils also form when guanidinium chloride is removed by dialysis of denatured Ab42CC at room temperature against 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl and 5 mM EDTA and a second dialysis for 4 to 6 hours in the same buffer without EDTA. Protofibrils of wild type Ab42 were identified in atomic force microscopy (AFM) images of an Ab42 aggregation reaction mixture. Ab42 monomer (,100 mM) in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl was kept at room temperature without shaking. A mixture of Ab42 protofibrils and smaller oligomers could be observed and distinguished in AFM images recorded after one day of incubation. The same solution was put in 37uC and shaking conditions for one more day in order to produce Ab42 fibrils for AFM imaging. Fibrils of wild type Ab40 and Ab42 for AFM and OC serum dot blot assays were prepared from similar mixtures that were subjected to 37uC and shaking to favor the formation of fibrils.equation solutions with a common (unimodal) frictional ratio. The partial specific volume of Ab42CC, and the buffer density and viscosity at 20uC, were calculated using the SEDNTERP program v 1.09 (Biomolecular Interaction Technologies Center, University of New Hampshire, Durham, NH; http://bitcwiki.sr.unh.edu/ index.php/Main_Page). Measured sedimentation coefficients were corrected to s20, w values (sedimentation coefficients in water at 20uC).Nanoparticle tracking analysis (NTA)Measurements were 23977191 performed with a NanoSight LM10-HS instrument (NanoSight, Amesbury, UK) equipped with an LM14 viewing unit using a 405 nm laser. Protofibrils of Ab42CC were diluted in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl to a final monomer concentration of 2 mM, and measurements were performed at 20uC for 30 s. The NanoSigh.
