ISSN:
0887-3585
Keywords:
ROP protein
;
4-α-helix-bundle
;
protein stability
;
cavity mutations
;
heat capacity
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
The structural and energetic perturbations caused by cavity-creating mutations (Leu-41 → Val and Leu-41 → Ala) in the dimeric 4-α-helical-bundle protein ROP have been characterized by CD spectroscopy and differential scanning calorimetry (DSC). Deconvolution of the CD spectra showed a decrease in α -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. ΔH0 values at the respective transition temperatures, T1/2 (71.0, 65.3, and 52.9°C at 0.5 mg/ml) decrease from 580 ± 20 to 461 ± 20 kJ/(mol of dimmer) and 335 ± 20 kJ/(mol of dimmer) for wildtype 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°C expressed by the standard Gibbs energies of denaturation, ΔGD0, are 71.7, 61.1, and 46.1 kJ/(mol of dimmer). The corresponding transition enthalpies have been obtained from extrapolation using the cpD(T)and cpN(T) functions. Their values at 25°C are 176.3, 101.9, and 141.7 kJ/(mol of dimmer) 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 ΔΔGD0 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 → Yal 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. © 1995 Wiley-Liss, Inc.
Additional Material:
9 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/prot.340230110
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