Major differences in stability and dimerization properties of two chimeric mutants of human stefins

Kenig, Manca; Jerala, Roman; Kroon-Zitko, L.; Türk, Vito; Žerovnik, Eva

doi:10.1002/1097-0134(20010301)42:4<512::aid-prot90>3.0.co;2-m

Cited by 9 publications

(2 citation statements)

References 48 publications

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“…2). Salt was added to 0.2 M to shield electrostatic interactions, which makes the data more comparable to those in GuHCl (Žerovnik et al 1992; Kenig et al 2001). The free energies of unfolding of −44 ± 5 kJ/mol obtained for both the G4R mutant and the wild‐type protein (Table 2) show that stefin B and the G4R mutant are stable proteins.…”

Section: Resultsmentioning

confidence: 60%

In vitro study of stability and amyloid‐fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

2005

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Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-a-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.

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Section: Resultsmentioning

confidence: 60%

In vitro study of stability and amyloid‐fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

2005

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“…The use of chimeras to evaluate contributions of protein segments to the stability and folding of the whole protein poses its own challenges and complications. Packing defects, resulting from creation of new interfaces between protein fragments, often reduce the overall thermodynamic stability (Kenig et al 2001). This masks any potential positive contribution of the studied fragment to the stability of the parent protein.…”

Section: Discussionmentioning

confidence: 99%

Contributions of folding cores to the thermostabilities of two ribonucleases H

2002

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To investigate the contribution of the folding cores to the thermodynamic stability of RNases H, we used rational design to create two chimeras composed of parts of a thermophilic and a mesophilic RNase H. Each chimera combines the folding core from one parent protein and the remaining parts of the other. Both chimeras form active, well-folded RNases H. Stability curves, based on CD-monitored chemical denaturations, show that the chimera with the thermophilic core is more stable, has a higher midpoint of thermal denaturation, and a lower change in heat capacity (⌬Cp) upon unfolding than the chimera with the mesophilic core. A possible explanation for the low ⌬Cp of both the parent thermophilic RNase H and the chimera with the thermophilic core is the residual structure in the denatured state. On the basis of the studied parameters, the chimera with the thermophilic core resembles a true thermophilic protein. Our results suggest that the folding core plays an essential role in conferring thermodynamic parameters to RNases H.

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Different propensity to form amyloid fibrils by two homologous proteins—Human stefins A and B: Searching for an explanation

et al. 2004

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By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.

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Major differences in stability and dimerization properties of two chimeric mutants of human stefins

Cited by 9 publications

References 48 publications

In vitro study of stability and amyloid‐fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

In vitro study of stability and amyloid‐fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

Contributions of folding cores to the thermostabilities of two ribonucleases H

Different propensity to form amyloid fibrils by two homologous proteins—Human stefins A and B: Searching for an explanation

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