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

Department of Biochemistry, Molecular and Structural Biology, Josef Stefan Institute, Ljubljana, Slovenia.
Amyloid (Impact Factor: 2.51). 10/2007; 14(3):237-47. DOI: 10.1080/13506120701461137
Source: PubMed

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 alpha-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.

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Available from: Rosemary A Staniforth, Jul 03, 2014
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    • "2,2,2-Trifluoroethanol (TFE) and its derivatives are commonly used as inhalation anesthetics. It is also used industrially as a solvent for nylon, as a dye and as an ingredient in anti-ulcer agents, anti-arrhythmic drugs etc. Toxicity of TFE has been confirmed on blood, male reproductive system, brain, upper respiratory tract and eyes (Zerovnik et al. 2007). It shifts the equilibrium from native toward denatured state and thus favoring aggregation and subsequent fibril formation. "
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    ABSTRACT: Aggregation of protein into insoluble intracellular complexes and inclusion bodies underlies the pathogenesis of human neurodegenerative diseases. Importance of cytochrome c (cyt c) arises from its involvement in apoptosis, sequence homology and for studying molecular evolution. A systemic investigation of polyethylene glycol (PEG) and trifluoroethanol (TFE) on the conformational stability of cyt c as a model hemeprotein was made using multi-methodological approach. Cyt c exists as molten globule (MG) at 60 % PEG-400 and 40 % TFE as confirmed by far-UV CD, attenuated total reflection Fourier transform infrared spectroscopy, Trp environment, 8-anilino-1-naphthalene-sulfonic acid (ANS) binding and blue shift in the soret band. Q-band splitting in MG states specifies conformational changes in the hydrophobic heme-binding pocket. Aggregates were detected at 90 % PEG-400 and 50 % TFE as confirmed by increase thioflavin T and ANS fluorescence and shift in Congo red absorbance. Detection of prefibrils and protofibrils at 90 % PEG-400 and 50 % TFE was possible after 72-h incubation. Single cell gel electrophoresis of prefibrils and protofibrils showed DNA damage confirming their toxicity and potential health hazards. Scanning electron microscopy and XRD analysis confirmed prefibrillar oligomers and protofibrils of cyt c.
    Amino Acids 04/2014; DOI:10.1007/s00726-014-1698-y · 3.65 Impact Factor
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    • "(U) has been shown previously to increase greatly and can comprise up to 1% of the sample (Zerovnik et al., 1998, 1999, 2007; Jelinska et al., 2011). Increasing the unfolding rate of dimers (red arrow) will allow a greater population of folded monomeric states to accumulate at any point in time: "
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    ABSTRACT: Unlike a number of amyloid-forming proteins, stefins, and in particular stefin B (cystatin B) form amyloids under conditions where the native state predominates. In order to trigger oligomerization processes, the stability of the protein needs to be compromised, favoring structural re-arrangement however, accelerating fibril formation is not a simple function of protein stability. We report here on how optimal conditions for amyloid formation lead to the destabilization of dimeric and tetrameric states of the protein in favor of the monomer. Small, highly localized structural changes can be mapped out that allow us to visualize directly areas of the protein which eventually become responsible for triggering amyloid formation. These regions of the protein overlap with the Cu (II)-binding sites which we identify here for the first time. We hypothesize that in vivo modulators of amyloid formation may act similarly to painstakingly optimized solvent conditions developed in vitro. We discuss these data in the light of current structural models of stefin B amyloid fibrils based on H-exchange data, where the detachment of the helical part and the extension of loops were observed.
    Frontiers in Molecular Neuroscience 10/2012; 5:94. DOI:10.3389/fnmol.2012.00094 · 4.08 Impact Factor
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    • "Amyloid fibril formation in vitro is also characteristics of stefins as of most proteins under suitable conditions [21] [22]. We have studied many aspects of this reaction [23] [24] [25] [26] and most were in accordance to other such systems, including cytotoxicity and membrane interaction of the prefibrillar oligomers [27] [28] [29]. Special feature of stefins and cystatins amyloid fibril formation is appearance of domain-swapped oligomers, dimers [11,27–30] and tetramers [9] [31]. "
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    ABSTRACT: The role of the aromatic residue at site 75 to protein stability, the mechanism of folding and the mechanism of amyloid-fibril formation were investigated for the human stefin B variant (bearing Y at site 31) and its point mutation H75W. With an aim to reveal the conformation at the cross-road between folding and aggregation, first, the kinetics of folding and oligomer formation by human stefin B(Y31) variant were studied. It was found to fold in three kinetic phases at pH 4.8 and 10% TFE; the pH and solvent conditions that transform the protein into amyloid fibrils at longer times. The same pH leads to the formation of native-like intermediate (known from previous studies of this variant), meaning that the process of folding and amyloid-fibril formation share the same structural intermediate, which is in this case native-like and dimeric. At pH 5.8 and 7.0 stefin B folded to the native state in four kinetic phases over two intermediates. In distinction, the mutant H75W did not fold to completion, ending in intermediate states at all pH values studied: 4.8, 5.8 and 7.0. At pH 4.8 and 5.8, the mutant folded in one kinetic phase to the intermediate of the "molten globule" type, which leads to the conclusion that its mechanism of folding differs from the one of the parent stefin B at the same pH. At pH 7.0 the mutant H75W folded in three kinetic phases to a native-like intermediate, analogous to folding of stefin B at pH 4.8.
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