Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-α-helical protein ROP: Thermal unfolding studies

C. Steif, H. J. Hinz, G. Cesareni

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Abstract

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, T( 1/2 ) (71.0, 65.3, and 52.9°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., 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, ΔG(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°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 ΔΔG(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.

Original languageEnglish
Pages (from-to)83-96
Number of pages14
JournalProteins: Structure, Function and Genetics
Volume23
Issue number1
DOIs
Publication statusPublished - 1995

Fingerprint

Dimers
Hot Temperature
Mutation
Specific heat
Enthalpy
Proteins
Dilatation and Curettage
Biochemistry
Denaturation
Transition Temperature
Differential Scanning Calorimetry
Deconvolution
Gibbs free energy
Point Mutation
Extrapolation
Conformations
Differential scanning calorimetry
Spectrum Analysis
Spectroscopy
Amino Acids

Keywords

  • 4-α-helix-bundle
  • cavity mutations
  • heat capacity
  • protein stability
  • ROP protein

ASJC Scopus subject areas

  • Genetics
  • Structural Biology
  • Biochemistry

Cite this

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title = "Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-α-helical protein ROP: Thermal unfolding studies",
abstract = "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, T( 1/2 ) (71.0, 65.3, and 52.9°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., 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, ΔG(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°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 ΔΔG(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.",
keywords = "4-α-helix-bundle, cavity mutations, heat capacity, protein stability, ROP protein",
author = "C. Steif and Hinz, {H. J.} and G. Cesareni",
year = "1995",
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TY - JOUR

T1 - Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-α-helical protein ROP

T2 - Thermal unfolding studies

AU - Steif, C.

AU - Hinz, H. J.

AU - Cesareni, G.

PY - 1995

Y1 - 1995

N2 - 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, T( 1/2 ) (71.0, 65.3, and 52.9°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., 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, ΔG(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°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 ΔΔG(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.

AB - 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, T( 1/2 ) (71.0, 65.3, and 52.9°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., 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, ΔG(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°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 ΔΔG(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.

KW - 4-α-helix-bundle

KW - cavity mutations

KW - heat capacity

KW - protein stability

KW - ROP protein

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