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

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: stef...

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Published in:	Proteins, structure, function, and bioinformatics Vol. 62; no. 4; pp. 918 - 927
Main Authors:	Kenig, Manca, Jenko-Kokalj, Saša, Tušek-Žnidarič, Magda, Pompe-Novak, Maruša, Gunčar, Gregor, Turk, Dušan, Waltho, Jonathan P., Staniforth, Rosemarie A., Avbelj, Franc, Žerovnik, Eva
Format:	Journal Article
Language:	English
Published:	Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-03-2006
Subjects:	Amyloid - chemistry Amyloid - genetics Amyloid - metabolism amyloid-fibril conformational disease cystatin Cystatin A Cystatin B Cystatins - chemistry Cystatins - genetics Cystatins - metabolism DNA Primers Guanidine Humans ionic interactions Models, Molecular Mutant Chimeric Proteins - chemistry Mutant Chimeric Proteins - genetics Polymerase Chain Reaction protein aggregation Protein Denaturation Protein Folding Protein Structure, Secondary stefin B
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Summary:	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 α‐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 β‐sheet of stefin B, and can be predicted reasonably well by calculating the β‐strand propensity of the denatured states. Proteins 2006. © 2005 Wiley‐Liss, Inc.
Bibliography:	ArticleID:PROT20812 The British Council and BBSRC (UK) Ministry for Higher Education and Science of the Republic of Slovenia - No. OB14P04SK; No. PO-0503 The Wellcome Trust ark:/67375/WNG-NKRNBRJR-Z The Royal Society istex:3343C4C8F7421D67AC833F1A4520C513E3970561 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0887-3585 1097-0134
DOI:	10.1002/prot.20812