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Amyloid
The Journal of Protein Folding Disorders
Volume 14, 2007 - Issue 3
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Original

Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology

, , , , , , , , , & show all
Pages 237-247 | Published online: 06 Jul 2009
 

Abstract

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all α-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.

Abbreviations
FSC=

forward scatter

SSC=

side scatter

C.R.=

Congo red

AFM=

atomic force microscopy

TEM=

transmission electron microscopy

GA=

glutaraldehyde

TFE=

2,2,2-trifluoroethanol

EtOH=

ethanol

HFIP=

1,1,1,3,3,3-hexafluoroisopropanol

CM-papain=

carboxy-methylated papain

PBS=

phosphate-buffered saline

FPLC=

fast protein liquid chromatography

Abbreviations
FSC=

forward scatter

SSC=

side scatter

C.R.=

Congo red

AFM=

atomic force microscopy

TEM=

transmission electron microscopy

GA=

glutaraldehyde

TFE=

2,2,2-trifluoroethanol

EtOH=

ethanol

HFIP=

1,1,1,3,3,3-hexafluoroisopropanol

CM-papain=

carboxy-methylated papain

PBS=

phosphate-buffered saline

FPLC=

fast protein liquid chromatography

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