Folding and amyloid‐fibril formation for a series of human stefins' chimeras: Any correlation?

Abstract

To study the influence of whole secondary structure elements to the process of folding and amyloid-fibril formation, chimeras of stefins have been prepared. GdnHCl denaturation curves and folding rates (chevron plots) have been analyzed based on a two-state mechanism. The order of stability is: stefin A > aAbbbb > bAbbbb > stefin B = aBaaaa > bBaaaa, where the make up of chimeric proteins is designated by small letters representing the source of individual strands (a for stefin A, b for stefin B) and a capital letter representing the source of the helix (A for stefin A and B for stefin B). Only the fast folding reactions were included in the analysis and it has been found that stefin B folds the fastest (657 s(-1)). Similarly, fast folders are the chimeric proteins aBaaaa and bBaaaa, both of which contain the alpha-helix of stefin B. Unfolding rates correlate very well with protein stability, with the slowest rate for the most stable protein, stefin A. Amyloid-fibril growth was measured for each protein by monitoring thioflavin T fluorescence and was visualized using electron microscopy. The propensity to form amyloid-fibrils is in the order: stefin B > bAbbbb > aAbbbb > bBaaaa > aBaaaa > stefin A. This order does not correlate with stability, or with the folding or unfolding rates. Instead, the propensity to fibrillize is related to selected parts of structure, such as the beta-sheet of stefin B, and can be predicted reasonably well by calculating the beta-strand propensity of the denatured states.

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation

“…Stefins are intracellular proteins present in the cytosol [14,15], including stefins A and B in humans [16] and stefins, A, B and C in bovidae [17,18]. Human stefin B [19,20,21,22,23], chimeric stefins [24] and cystatin C [25,26] have been used as suitable model proteins to study protein folding and amyloid fibril formation. Human stefin B is a small globular protein consisting of 98 amino acids with no disulfide bonds; its native sequence possesses a free Cys residue at position 3.…”

Mapping local structural perturbations in the native state of stefin B (cystatin B) under amyloid forming conditions

“…The mechanism of amyloid fibril formation by human stefins A and B and their chimeras has been studied extensively by our groups (Kenig et al, 2006; Jelinska et al, 2011; Žerovnik et al, 2011). While stefin B amyloids have not been observed directly in vivo , mutations in the gene encoding stefin B cause a progressive form of Myoclonus Epilepsy (Unverricht-Lundborg disease or EPM 1) and it has been proposed that these mutations correlate with their aggregation behavior in vitro (reviewed in Polajnar et al, 2012).…”

The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones

“…A similar lack of correlation between stability and fibrillation was observed with wild-type and chimeric human stefins. 46 The propensity of the histones to form fibrils does correlate with solubility. It was previously known that archaeal histones are highly soluble; during purification, hMfB and hPyA1 remain in solution at 60-70% ammonium sulfate at neutral pH.…”