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 ol-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-0 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 wildtype ROP (Steif, C., Weber, P., Hint, H.-J., Flossdorf, J., Cesareni, G., Kokkinidis, M. Biochemistry 32:3867-3876, 1993), L(41)V, and L(41)A, respectively. The conformational stabilities at 25 degrees C expressed by the standard Gibbs energies of denaturation, Delta G(D)(0), are 71.7, 61.1, and 46.1 kJ/(mol of dimer). The corresponding transition enthalpies have been obtained from extrapolation using the c(p)(D)(T) and c(p)(N)(T) functions. Their values at 25 degrees C are 176.3, 101.9, and 141.7 kJ/(mol of dimer) for wild-type ROP, L(41)V, and L(41)A, 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 G(D)(0) 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. (C) 1995 Wiley-Liss, Inc.