Enthalpic barriers dominate the folding and unfolding of the human Cu, Zn superoxide dismutase monomer

Abstract

The rate-limiting step in the formation of the native dimeric state of human Cu, Zn superoxide dismutase (SOD1) is a very slow monomer folding reaction that governs the lifetime of its unfolded state. Mutations at dozens of sites in SOD1 are known to cause a fatal motor neuron disease, amyotrophic lateral sclerosis, and recent experiments implicate the unfolded state as a source of soluble oligomers and histologically observable aggregates thought to be responsible for toxicity. To determine the thermodynamic properties of the transition state ensemble (TSE) limiting the folding of this high-contact-order β-sandwich motif, we performed a combined thermal/urea denaturation thermodynamic/kinetic analysis. The barriers to folding and unfolding are dominated by the activation enthalpy at 298 K and neutral pH; the activation entropy is favorable and reduces the barrier height for both reactions. The absence of secondary structure formation or large-scale chain collapse prior to crossing the barrier for folding led to the conclusion that dehydration of nonpolar surfaces in the TSE is responsible for the large and positive activation enthalpy. Although the activation entropy favors the folding reaction, the transition from the unfolded state to the native state is entropically disfavored at 298 K. The opposing entropic contributions to the free energies of the TSE and the native state during folding provide insights into structural properties of the TSE. The results also imply a crucial role for water in governing the productive folding reaction and enhancing the propensity for the aggregation of SOD1.

Figure 1. Crystal structure and topology of SOD1

A) SOD1 is a dimeric β-sandwich protein consisting of eight anti-parallel β-stands supporting two catalytic loops (PDB: 2C9V). The electrostatic loop is depicted in green, while the Zn-binding loop is depicted in cyan. Each monomeric subunit also contains a Zn (blue sphere) and Cu ion (orange sphere), which were not present for this study, as well as an intramolecular disulfide bond (yellow). The free cysteines, C6/C111 (red), were replaced with alanine and serine respectively. Additionally, a pair of glutamic acid residues was introduced in the dimer interface, replacing F50/G51 (violet), in order to obtain the obligate monomer, mSOD1*. B) The topology of SOD1 has the immunoglobulin fold. It is made up of two β-sheetsconsisting of strands β1β2β3β6 and β5β4β7β8, and contains a Greek-key motif. The loops have been labeled with sequential Roman numerals.

Figure 1. Crystal structure and topology of SOD1

A) SOD1 is a dimeric β-sandwich protein consisting of eight anti-parallel β-stands supporting two catalytic loops (PDB: 2C9V). The electrostatic loop is depicted in green, while the Zn-binding loop is depicted in cyan. Each monomeric subunit also contains a Zn (blue sphere) and Cu ion (orange sphere), which were not present for this study, as well as an intramolecular disulfide bond (yellow). The free cysteines, C6/C111 (red), were replaced with alanine and serine respectively. Additionally, a pair of glutamic acid residues was introduced in the dimer interface, replacing F50/G51 (violet), in order to obtain the obligate monomer, mSOD1*. B) The topology of SOD1 has the immunoglobulin fold. It is made up of two β-sheetsconsisting of strands β1β2β3β6 and β5β4β7β8, and contains a Greek-key motif. The loops have been labeled with sequential Roman numerals.

Figure 2. Temperature dependence of the ΔG⁰, m-values and m^‡-values

A) The free energy of folding in the absence of denaturant and B) the m-values of folding are shown as a function of temperature as derived from kinetic (open diamonds) and equilibrium (filled circles) experiments. C) The refolding (open squares), and unfolding (filled squares) kinetic m^‡-values are shown as a function of temperature. The error bars represent the standard deviation of the fit. The solid linesin panel A represent the best fit to the Gibbs-Helmholtz equation (Equation 1).

Figure 3. Temperature dependence of mSOD1* folding kinetics

The folding (open symbols) and unfolding (filled symbols) relaxation times of mSOD1*, as a function of denaturant concentration, are shown at every 5 K. A) 283 K (circles), 288 K (squares), 293 K (up triangle), 298 K (down triangle), 303 K (diamond), 308 K (hexagon), and 313 K (up triangle). B) 285.5 K (circles), 290.5 K (squares), 295.5 K (up triangle), 300.5 K (down triangle), 305.5 K (diamond), and 310.5 K (hexagon).

Figure 4. Fit of the kinetic data to the Kramers model

The natural log of the folding (open circles) and unfolding (filled circles) rate constants divided by the temperature were plotted as a function of the inverse of the temperature and fit to the Kramers model as described in the Results to obtain the thermodynamic parameters of the transition state, ΔS^0‡, ΔH^0‡ and $\mathrm{\Delta }{C}_p^{0^{‡}}$ (Table 1).

Figure 5. Reaction Coordinate Diagram for mSOD1*

The enthalpic, ΔH⁰, (filled triangle) and entropic, −TΔS⁰ (open circle) contributions to the free energies (open square) of the M, TSE, and U states at the standard state of 298 K and using the M state as the reference state are shown. The placement of the TSE and U states relative to M was based on m^‡-values and m-values extracted from the kinetic folding data analysis.