Subunit interactions provide a significant contribution to the stability of the dimeric four-.alpha.-helical-bundle protein ROP

Abstract

Detailed thermodynamic and spectroscopic studies were carried out on the ColE1-ROP protein in order to establish a quantitative basis for the contribution of noncovalent interactions to the stability of four-helix-bundle proteins. The energetics of both heat- and GdnHCl-induced denaturation were measured by differential scanning microcalorimetry (DSC) and/or by following the change in circular dichroism in the far-UV range. Sedimentation equilibrium analyses were performed to characterize the state of aggregation of the protein. No intermediate species could be detected during thermal unfolding of the dimer in the absence of GdnHCl. Under these conditions ROP unfolding exhibits a strict two-state behavior. The thermodynamic parameters for the reaction N2<->2D are delta HD = 580 +/- 20 kJ.(mol of dimer)-1, delta Cp = 10.3 +/- 1.3 kJ.(mol of dimer)-1.K-1, and Tm = 71.0 +/- 0.5 degrees C. The corresponding Gibbs energy change of unfolding is delta GD degree = 71.7 kJ.(mol of dimer)-1 at 25 degrees C and pH 6. In the presence of 2.5 M GdnHCl, however, ROP dissociates into monomers at elevated temperatures, as the loss of the concentration dependence of Tm and the decreased molecular weight demonstrate. The corresponding transition parameters are delta HD (2.5 M GdnHCl) = 130 +/- 10 kJ.(mol of monomer)-1 and Tm = 51.6 +/- 0.3 degrees C. Isothermal unfolding studies at 19 degrees C using GdnHCl as denaturant yielded a Gibbs energy change of unfolding of 22.4 kJ.(mol of monomer)-1. This extrapolated value is 38% lower than the corresponding delta GD degree value of 35.85 kJ.(mol of monomer)-1 calculated from thermal unfolding for the monomer in the absence of GdnHCl, where the protein is known to be a dimer. These results suggest that subunit interactions are an important source of stabilization of the native four-helix-bundle structure of ROP.

Redesigning the hydrophobic core of a four‐helix‐bundle protein

“…See Table 2. We should note that our results for wild-type Ropl 1 are in fair agreement with those published by Steif et al (1993), who used a buffer of much lower ionic strength than we did. The greatest difference is in ACD, which, as mentioned above, when eval- matic difference in the rates of folding and unfolding of the wild-type and repacked proteins suggests that the hydrophobic core of Rop plays a critical role in the rate-determining step of folding.…”

“…This observation provides an explanation for the results of Steif et al (1993), who reported, using 24-h equilibration times, that there was hysteresis between the GuHC1-induced folding and unfolding curves for wild-type Rop. They reported the midpoint of the unfolding transition as approximately 5.3 M and of the folding as approximately 2.5 M, at 19 "C with protein concentrations of 0.3 mg/mL.…”

Characterization of a new four‐chain coiled‐coil: Influence of chain length on stability

“…("""_) , and monomer-dimer (----) equilibrium schemes 'Steif et al (1993) ' Chmielewski and Lipton (1994) ..…”

Biophysical Characterization of a β-Peptide Bundle: Comparison to Natural Proteins

“…The integrated calorimetric unfolding enthalpy (∆H Cal ) for Zwit-1F is 7.2 cal‚g -1 , within the range observed for natural globular proteins (5.2-11.8 cal‚g -1 ), 19,20 but somewhat lower than GCN4 (7.7 cal‚g -1 ) 21 and ROP (9.5 cal‚g -1 ). 17 The NMR spectra of many well-folded natural and designed proteins are characterized by differentiated amide resonances and slow hydrogen/deuterium exchange. 22 The amide N-H resonances in the 1 H spectrum of Zwit-1F, under conditions where the sample is 97% octameric, span 1.4 ppm ( Figure 3A).…”