Differences in aggregation properties of three site-specific mutants of recombinant human stefin B
| Autor: | Magda Tus̆ek, Eva Zerovnik, Maruša Pompe-Novak, L. Kroon-Zitko, Manca Kenig, Aida Krijes̆torac, Selma Berbić |
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| Jazyk: | angličtina |
| Rok vydání: | 2004 |
| Předmět: |
Models
Molecular Circular dichroism Amyloid Protein Folding Databases Factual Dimer Mutant Plasma protein binding Biochemistry Article Anilino Naphthalenesulfonates Protein Structure Secondary law.invention chemistry.chemical_compound law Escherichia coli Humans Point Mutation Urea Cystatin B Binding site Molecular Biology Fluorescent Dyes Binding Sites Circular Dichroism Temperature Genetic Variation Chromatography Ion Exchange Cystatins Recombinant Proteins Protein Structure Tertiary chemistry Mutation Recombinant DNA Chromatography Gel Mutagenesis Site-Directed Protein folding Dimerization Protein Binding |
| Popis: | We describe expression, purification, and characterization of three site-specific mutants of recombinant human stefin B: H75W, P36G, and P79S. The far- and near-UV CD spectra have shown that they have similar secondary and tertiary structures to the parent protein. The elution on gel-filtration suggests that recombinant human stefin B and the P36G variant are predominantly monomers, whereas the P79S variant is a dimer. ANS dye binding, reflecting exposed hydrophobic patches, is highest for the P36G variant, both at pH 5 and 3. ANS dye binding also is increased for stefin B and the other two variants at pH 3. Under the chosen conditions the highest tendency to form amyloid fibrils has been shown for the recombinant human stefin B. The P79S variant demonstrates a longer lag phase and a lower rate of fibril formation, while the P36G variant is most prone to amorphous aggregation. This was demonstrated by ThT fluorescence as a function of time and by transmission electron microscopy. |
| Databáze: | OpenAIRE |
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