Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-alpha-helical protein ROP: thermal unfolding studies.
The structural and energetic perturbations caused by cavity-creating mutations (Leu-41-->Val and Leu-41-->Ala) in the dimeric 4-alpha-helical-bundle protein ROP have been characterized by CD spectroscopy and differential scanning calorimetry (DSC). Deconvolution of the CD spectra showed a decrease in alpha-helicity as a result of the amino acid exchanges that follows qualitatively the overall decrease in conformational stability. Transition enthalpies are sensitive probes of the energetic change associated with point mutations. delta H zero values at the respective transition temperatures, T 1/2 (71.0, 65.3, and 52.9 degrees C at 0.5 mg/ml) decrease from 580 +/- 20 to 461 +/- 20 kJ/(mol of dimer) and 335 +/- 20 kJ/(mol of dimer) for wild-type ROP (Steif, C., Weber, P., Hinz, H.-J., Flossdorf, J., Cesareni, G., Kokkinidis, M. Biochemistry 32:3867-3876, 1993), L41V, and L41A, respectively. The conformational stabilities at 25 degrees C expressed by the standard Gibbs energies of denaturation, delta GzeroD, are 71.7, 61.1, and 46.1 kJ/(mol of dimer). The corresponding transition enthalpies have been obtained from extrapolation using the cDp(T) and cNp(T) functions. Their values at 25 degrees C are 176.3, 101.9, and 141.7 kJ/(mol of dimer) for wild-type ROP, L41V, and L41A, respectively. When the stability perturbation resulting from the cavity creating mutations is referred to the exchange of 1 mol of CH2 group, the average delta delta GzeroD value is -5.0 +/- 1 kJ/(mol of CH2 group). This decrease in conformation stability suggests that dimeric ROP exhibits the same susceptibility to Leu-->Val and Leu-->Ala exchanges as small monomeric proteins. Careful determinations of the partial specific heat capacities of wild-type and mutated protein solutions suggest that the mutational effects are predominantly manifested in the native rather than the unfolded state.
Institut für Physikalische Chemie, Westfälischen Wilhelms-Universität, Münster, Germany.
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D000409:Alanine; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004735:Energy Transfer; D004795:Enzyme Stability; D006358:Hot Temperature; D007930:Leucine; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012984:Software; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D014633:Valine
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C054243:Rop protein, ColE1 plasmid; D007930:Leucine; D014633:Valine; D000409:Alanine