Table 1.
Mutations.
Figure 1.
Localization of side chains affected by mutations and residual enzyme function of MCAD variants.
(A) Mutations in the ACADM gene map to side chains (red) of all regions of the MCAD monomer. The mature MCAD protein is shown and numbering of side chains refers to the primary sequence after cleavage of an N-terminal 25 amino acids mitochondrial targeting peptide (see Table 1). Side chains, FAD cofactor (yellow) and the substrate analogue 3-thiaoctanoyl-CoA (orange) are shown as stick models. (B, C) Structural localization of affected side-chains at the surface of subunit A (gray) relative to the other subunits (B, green; C pink; D, blue) in the MCAD tetramer. (D) Surface view of the MCAD tetramer with 3-thiaoctanoyl-CoA (orange) bound to the substrate binding site and the positioning of FAD (yellow) relative to subunits A (gray), B (green), and D (blue). (E) Relative enzyme activity of wild-type and variant MCAD proteins. Specific activity was determined using purified recombinant protein [16] and residual activity was measured upon expression in COS-7 cells. Data are given as means and SD of three replicates.
Figure 2.
Missense mutations induce conformational alterations of variant MCAD proteins.
(A) The composition of secondary structure elements of wild-type and variant MCAD in the absence of thermal stress was determined by circular dichroism applying the CD spectra deconvolution software CDNN [23]. The spectra showed typical secondary structure characteristics of proteins with predominating α-helical content. (B) Hydrophobicity of wild-type and variant MCAD proteins in the absence of thermal stress was determined using the fluorescent dye ANS. An increase in the fluorescence signal points to increased binding of the ANS dye to hydrophobic groups. Data represent means and SEM of three independent experiments. (C) Structural integrity of wild-type and variant MCAD proteins in the absence of thermal stress was determined by intrinsic FAD fluorescence. An increase in the fluorescence signal points to a release of FAD from the MCAD protein. Data represent means and SEM of three independent experiments. (B, C) Data are grouped according to the domain affected by mutations (αDN, N-terminal α-helical domain; βD, β-sheet domain; αDC, C-terminal α-helical domain).
Table 2.
Secondary structure analysis (CD spectroscopy).
Figure 3.
Mutations of the middle β-domain of the MCAD protein are associated with accelerated heat-induced unfolding.
(A) Thermal stress-induced denaturation monitored by circular dichroism (CD) probing loss of secondary structure. Denaturation profiles of the fractional change of the α-helical secondary structures of wild-type and variant MCAD were measured at 208 nm. Data are given as non-linear fit of the mean of three independent experiments where 1 represents the native state and 0 the unfolded state. (B) Thermal stress-induced denaturation monitored by FAD differential scanning fluorimetry (DSF) probing FAD binding capacity. The temperature dependent release of intrinsic FAD of wild-type and variant MCAD was measured at 530 nm (excitation 450 nm). Data are given as non-linear fit of the mean of three independent experiments where 0 represents the native state and 1 the unfolded state. (C) Thermal stress-induced denaturation monitored by ANS-DSF probing global unfolding. Unfolding profiles of wild-type and variant MCAD were determined as changes in ANS fluorescence emission at 450 nm (excitation 395 nm). All unfolding patterns revealed a biphasic unfolding mechanism with a distinct low- and high-temperature transition, depicted in two red lines in the unfolding curve of wild-type MCAD. Data are grouped according to the domain affected by mutations (αDN, N-terminal α-helical domain; βD, β-sheet domain; αDC, C-terminal α-helical domain) and given as mean of three independent experiments where 0 represents the native state and 1 the unfolded state.
Table 3.
Mean transition midpoints of thermal denaturation (CD spectroscopy, FAD fluorescence, ANS fluorescence).
Figure 4.
MCAD variants are prone to aggregation.
Right angle light scattering experiments probing thermal stress-induced aggregation. The intensity of the scattered light as a function of increasing temperature was determined at 335 nm (excitation 330 nm) for variants arising from mutations mapping to the N-terminal α-domain (αDN), variants arising from mutations mapping to the middle β-domain (βD), and variants with amino acid substitutions in the C-terminal α-domain (αDC).
Figure 5.
Kinetics of thermal inactivation.
(A) Kinetics of thermal inactivation experiments probing dynamics of heat-induced loss of function. Wild-type and variant MCAD proteins were incubated at indicated temperatures and residual activity was plotted as a function of incubation time. (B) Arrhenius Plots resulting from kinetic constants k of time-dependent thermal denaturation experiments. (C) Activation energies (EA) calculated from Arrhenius Plots reflect the energy barrier between the conformational state with full residual activity and the non-active state after application of thermal stress.
Figure 6.
Molecular phenotypes of thermal, kinetic, and conformational stability.
Data derived from experiments depicted in Figures 2, 3, and 5 were combined and visualized as 3D plot. Thermal stability (y-axis) refers to midpoints of thermal denaturation determined by CD spectroscopy, FAD-DSF, and ANS-DSF. Kinetic stability (x-axis) refers to the EA determined by thermal inactivation experiments. Conformational stability (z-axis) refers to secondary structure (CD spectra), hydrophobicity (ANS fluorescence), and FAD binding capacity (intrinsic FAD fluorescence) in the absence of heat-induced stress.