Differences in aggregation properties of three site-specific mutants of recombinant human stefin B.
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.
Kenig
Manca
M
Department of Biochemistry and Molecular Biology, Joef Stefan Institute, 1000 Ljubljana, Slovenia.
Berbić
Selma
S
Krijestorac
Aida
A
Kroon-Zitko
Louise
L
Tusek
Magda
M
Pompe-Novak
Marusa
M
Zerovnik
Eva
E
D000682:Amyloid / Q000378:metabolism / Q000648:ultrastructure; D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D055313:Cystatin B; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism*; D016208:Databases, Factual; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D014644:Genetic Variation; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D000682:Amyloid; D000817:Anilino Naphthalenesulfonates; C524994:CSTB protein, human; D015891:Cystatins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D055313:Cystatin B; D014508:Urea