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Differences in aggregation properties of three site-specific mutants of recombinant human stefin B

Manca Kenig^1,4, Selma Berbi^2,4, Aida Krijetorac², Louise Kroon-itko¹, Magda Tuek³, Marua Pompe-Novak³ and Eva erovnik¹

¹ Department of Biochemistry and Molecular Biology, Joef Stefan Institute, 1000 Ljubljana, Slovenia
² Department of Biochemistry, Medical Faculty of Tuzla, University of Tuzla, 75000 Tuzla, Bosnia and Herzegovina
³ Department of Plant Physiology and Biotechnology, National Institute of Biology, 1000 Ljubljana, Slovenia

Reprint requests to: Eva erovnik, Department of Biochemistry and Molecular Biology, Joef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; e-mail: eva.zerovnik{at}ijs.si; fax: 386-1-257-35-94.

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.

Keywords: amyloid-fibrils; circular dichroism (CD); cystatins; conformational disease; domain-swapped dimer; proline mutants; protein folding

Abbreviations: ANS, 1, anilino-naphthalene 8-sulfonate • ES MS, electrospray mass spectrometry • IPTG, isopropyl-ß-D-thiogalactopyranoside • SEC, size-exclusion chromatography • TEM, transmission electron microscopy • TFE, 2,2,2-trifluoroethanol • ThT, thioflavin T

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