Thermal unfolding of two designed monomeric λ Cro repressor variants

  • Fabian H
  • Gast K
  • Filimonov V
 et al. 
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Abstract

The thermal unfolding of the wild-type λ Cro repressor and of two designed variants, Cro K56-[DGEVK] and Cro K56-[DGEVK] Q16L, was studied by Fourier transform infrared spectroscopy and dynamic light scattering. The engineered Cro K56-[DGEVK] monomer has five additional amino acids inserted after position 56 of the wild-type sequence, while the K56-[DGEVK] Q16L variant differs only in one position (Gln-16 to Leu substitution) from the Cro K56-[DGEVK] sequence. The temperature dependence of selected protein backbone infrared 'marker' bands revealed that Cro K56-[DGEVK] is slightly more stable than the wild-type protein, while the replacement of Gln-16 by Leu increases the thermal transition temperature by ∼20 °C. Moreover, thermal unfolding of the two Cro variants was found to proceed through equilibrium unfolding intermediates and to involve the formation of oligomers. The first thermal transition of Cro K56-[DGEVK] involves the melting of major parts of its native secondary structure and is accompanied by the formation of dimers and non-native β-sheet structures. These structures unfold during a second transition at higher temperatures, accompanied by the dissociation of the dimers. In contrast to the Cro K56-[DGEVK] protein, the intermediate state of the Cro K56-[DGEVK] Q16L variant is less well defined, and involves the formation of oligomers of different size. © 2005 Elsevier B.V. All rights reserved.

Author-supplied keywords

  • Oligomer
  • Thermal unfolding
  • λ Cro repressor

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Authors

  • Heinz Fabian

  • Klaus Gast

  • Vladimir V. Filimonov

  • Dmitry F. Zamyatkin

  • Vladimir V. Rogov

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