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Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-alpha-helical protein ROP: thermal unfolding studies.
Proteins. 1995 Sep; 23(1):83-96.P

Abstract

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.

Authors+Show Affiliations

Institut für Physikalische Chemie, Westfälischen Wilhelms-Universität, Münster, Germany.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8539253

Citation

Steif, C, et al. "Effects of Cavity-creating Mutations On Conformational Stability and Structure of the Dimeric 4-alpha-helical Protein ROP: Thermal Unfolding Studies." Proteins, vol. 23, no. 1, 1995, pp. 83-96.
Steif C, Hinz HJ, Cesareni G. Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-alpha-helical protein ROP: thermal unfolding studies. Proteins. 1995;23(1):83-96.
Steif, C., Hinz, H. J., & Cesareni, G. (1995). Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-alpha-helical protein ROP: thermal unfolding studies. Proteins, 23(1), 83-96.
Steif C, Hinz HJ, Cesareni G. Effects of Cavity-creating Mutations On Conformational Stability and Structure of the Dimeric 4-alpha-helical Protein ROP: Thermal Unfolding Studies. Proteins. 1995;23(1):83-96. PubMed PMID: 8539253.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-alpha-helical protein ROP: thermal unfolding studies. AU - Steif,C, AU - Hinz,H J, AU - Cesareni,G, PY - 1995/9/1/pubmed PY - 1995/9/1/medline PY - 1995/9/1/entrez SP - 83 EP - 96 JF - Proteins JO - Proteins VL - 23 IS - 1 N2 - 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. SN - 0887-3585 UR - https://www.unboundmedicine.com/medline/citation/8539253/Effects_of_cavity_creating_mutations_on_conformational_stability_and_structure_of_the_dimeric_4_alpha_helical_protein_ROP:_thermal_unfolding_studies_ L2 - https://doi.org/10.1002/prot.340230110 DB - PRIME DP - Unbound Medicine ER -