Introduction of a proline residue into position 31 of the loop of the dimeric 4-α-helical protein ROP causes a drastic destabilization

Kirsten Peters, Hans Jürgen Hinz, Gianni Cesareni

Research output: Contribution to journalArticle

Abstract

The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-α-helical-bundle protein ROP causes a reduction in the α-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters. The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, ΔG(D)0 (buffer, 25°C), has been determined to be 43 kJ (mol dimer)-1. The corresponding ROPwt value is 72 kJ (mol dimer)-1. The extrapolated ΔG(D)0 values obtained from urea and GdmHCl un- and refolding studies are identical within error limits. Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters. At T(1/2) = 43 °C(protein) = 0.05 mg ml-1) the ROP A31p mutant is characterized by ΔH(v.)0(H.) = 272 kJ (mol dimer)-1, ΔC(p) = 7.2 kJ (mol dimer)-1 K-1, ΔS0 = 762 J (mol dimer)-1 K-1. These parameters are only approximately 50% as large as the corresponding values of ROPwt. We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure. This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm. All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability.

Original languageEnglish
Pages (from-to)1141-1152
Number of pages12
JournalBiological Chemistry
Volume378
Issue number10
Publication statusPublished - Oct 1997

Keywords

  • 4-α-helix-bundle
  • Loop mutation
  • Proline
  • Protein stability
  • ROP protein

ASJC Scopus subject areas

  • Biochemistry

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