Conformational stability and thermodynamic characterization of the lipoic acid bearing domain of human mitochondrial branched chain alpha-ketoacid dehydrogenase

Abstract

The lipoic acid bearing domain (hbLBD) of human mitochondrial branched chain alpha-ketoacid dehydrogenase (BCKD) plays important role of substrate channeling in oxidative decarboxylation of the branched chain alpha-ketoacids. Recently hbLBD has been found to follow two-step folding mechanism without detectable presence of stable or kinetic intermediates. The present study describes the conformational stability underlying the folding of this small beta-barrel domain. Thermal denaturation in presence of urea and isothermal urea denaturation titrations are used to evaluate various thermodynamic parameters defining the equilibrium unfolding. The linear extrapolation model successfully describes the two-step; native state <-->denatured state unfolding transition of hbLBD. The average temperature of maximum stability of hbLBD is estimated as 295.6 +/- 0.9 K. Cold denaturation of hbLBD is also predicted and discussed.

Figure 1.

Ribbon representation of the structure of hbLBD. The domain is a flattened β barrel comprising two four-stranded β-sheets. The residues involved in missense mutation are also depicted. The amino acids are numbered according to the original sequence, which is one residue less than the sequence in Protein Data Bank file (PDB code 1k8m). This figure is created with MOLMOL version 2K.2.0 (Koradi et al. 1996).

Figure 2.

Effect of thermal denaturation on CD spectra. The hbLBD spectra (pH = 7.6 ± 0.1) collected at 280K (bold dotted line), 295K (bold solid line), 325K (dashed line), 335K (“dash-dot-dot-dash” line), 340K (solid line), 345K (dotted line), 350K (“dash-dot-dash” line), and 355K (bold dashed line) at 0.5-nm resolution are shown.

Figure 3.

(A) Isothermal urea denaturation titration curves. The urea titration of hbLBD (pH = 7.6 ± 0.1) at 275 (•), 280K (▾), 285K (○), 290K (▪), 295K (*), 300K (▴), 305K (⋄), 310K (□), 315K (▴), and 320K (♦) is shown, with solid lines representing nonlinear regression fit to equation 7 assuming linear extrapolation model. Effect of temperature on free energy change in absence of denaturant (ΔG_H2O) (B); half denaturation urea concentration, C_mid (C); and the m value (D). All parameters are calculated from nonlinear regression analysis for equation 7 as shown in A, and the numerical results are tabulated in the Supplemental Material.

Figure 4.

Thermal denaturation profiles. The effect of temperature on hbLBD in various urea concentrations at pH = 7.6 ± 0.1 as monitored by far-UV CD at 228 nm. Thermal scans in the absence (*) and presence of 0.25 M (▪), 0.5 M (○), 0.75 M (▾), 1.0 M (▴), 1.25 M (•), 1.5 M (⋄,) 1.75 M (▴), 2.0 M (□), 2.25 M (▿), 2.5 M (♦), 2.75 M (+), 3 M (×), 3.25 M (▵), 3.5 M (□), 3.75 M (○), and 4 M (▿) urea are shown. The solid sigmoids indicate nonlinear regression fit to equation 13.

Figure 5.

The stability curves. Effect of temperature on conformational free energy at different urea concentrations. The urea concentration in various curves is 0.0 M (*), 0.25 M (▪), 0.5 M (○), 0.75 M (▾), 1.0 M (▵), 1.25 M (•), 1.5 M (⋄), 1.75 M (▴), 2.0 M (□), 2.25 M (▿), 2.5 M (♦), 2.75 M (+), 3 M (×), 3.25 M (▵), 3.5 M (□), 3.75 M (○), and 4 M (▿). The dotted line is used to distinguish data from urea titration and thermal denaturation studies. Majority of data from titration experiments (ΔG_H2O and ΔG_D) is on left and above the dotted line, and data from thermal denaturation (ΔG_T) are shown on right and below the dotted line. The perfect agreement in these two data sets validates equilibrium measurements. The solid curves are regression fit to equation 13.

Figure 6.

Plot of ΔHG against TG. The linear relationship in ΔHG and TG with slope indicating the apparent ΔCp of 1.77 ± 0.05 kcal/mole/K.

Figure 7.

Effect of urea concentration on conformational stability, ΔG_S of hbLBD. The linear relationship with the Y-axis intercept of 5.69 ± 0.02 and slope of −1.10 ± 0.01 are in good agreement with results from titration experiments, summarized in Figure 3 ▶ and the Supplemental Material. The error bars are calculated by solving error propagation equation.