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Differences in aggregation properties of three site‐specific mutants of recombinant human stefin B
Author(s) -
Kenig Manca,
Berbić Selma,
Krijes̆torac Aida,
KroonZ̆itko Louise,
Tus̆ek Magda,
PompeNovak Marus̆a,
Z̆erovnik Eva
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03270904
Subject(s) - recombinant dna , dimer , chemistry , monomer , mutant , size exclusion chromatography , biophysics , protein aggregation , fluorescence , amyloid (mycology) , fibril , biochemistry , biology , gene , polymer , inorganic chemistry , physics , organic chemistry , quantum mechanics , enzyme
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.