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The mechanism of amyloid‐fibril formation by stefin B: Temperature and protein concentration dependence of the rates
Author(s) -
Škerget Katja,
Vilfan Andrej,
PompeNovak Maruša,
Turk Vito,
Waltho Jonathan P.,
Turk Dušan,
Žerovnik Eva
Publication year - 2008
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.22156
Subject(s) - chemistry , nucleation , kinetics , thioflavin , fibril , enthalpy , tetramer , globular protein , isomerization , temperature jump , crystallography , protein folding , rate determining step , biophysics , thermodynamics , biochemistry , enzyme , medicine , physics , disease , organic chemistry , pathology , quantum mechanics , biology , catalysis , alzheimer's disease
Cystatins, a family of structurally related cysteine proteinase inhibitors, have proved to be useful model system to study amyloidogenesis. We have extended previous studies of the kinetics of amyloid‐fibril formation by human stefin B (cystatin B) and some of its mutants, and proposed an improved model for the reaction. Overall, the observed kinetics follow the nucleation and growth behavior observed for many other amyloidogenic proteins. The minimal kinetic scheme that best fits measurements of changes in CD and thioflavin T fluorescence as a function of protein concentration and temperature includes nucleation (modeled as N I irreversible transitions with equivalent rates ( k I ), which fitted with N I = 64), fibril growth and nonproductive oligomerization, best explained by an off‐pathway state with a rate‐limiting escape rate. Three energies of activation were derived from global fitting to the minimal kinetic scheme, and independently through the fitting of the individual component rates. Nucleation was found to be a first‐order process within an oligomeric species with an enthalpy of activation of 55 ± 4 kcal mol −1 . Fibril growth was a second‐order process with an enthalpy of activation (27 ± 5 kcal mol −1 ), which is indistinguishable from that of tetramer formation by cystatins, which involves limited conformational changes including proline trans to cis isomerization. The highest enthalpy of activation (95 ± 5 kcal mol −1 at 35°C), characteristic of a substantial degree of unfolding as observed prior to domain‐swapping reactions, equated with the escape rate of the off‐pathway oligomeric state. Proteins 2009. © 2008 Wiley‐Liss, Inc.