Metal-free ALS variants of dimeric human Cu,Zn-superoxide dismutase have enhanced populations of monomeric species.

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.

Mapping the folding free energy surface for metal-free human Cu,Zn superoxide dismutase.

Mutations at many different sites in the gene encoding human Cu,Zn superoxide dismutase (SOD) are known to be causative agents in amyotrophic lateral sclerosis (ALS). One explanation for the molecular basis of this pathology is the aggregation of marginally soluble, partially structured states whose populations are enhanced in the protein variants. As a benchmark for testing this hypothesis, the equilibrium and kinetic properties of the reversible folding reaction of a metal-free variant of SOD were investigated. Reversibility was achieved by replacing the two non-essential cysteine residues with non-oxidizable analogs, C6A/C111S, to produce apo-AS-SOD. The metal-free pseudo-wild-type protein is folded and dimeric in the absence of chemical denaturants, and its equilibrium folding behavior is well described by an apparent two-state mechanism involving the unfolded monomer and the native dimer. The apparent free energy of folding in the absence of denaturant and at standard state is -20.37(+/- 1.04) kcal (mol dimer)(-1). A global analysis of circular dichroism kinetic traces for both unfolding and refolding reactions, combined with results from small angle X-ray scattering and time-resolved fluorescence anisotropy measurements, supports a sequential mechanism involving the unfolded monomer, a folded monomeric intermediate, and the native dimer. The rate-limiting monomer folding reaction is followed by a near diffusion-limited self-association reaction to form the native dimer. The relative population of the folded monomeric intermediate is predicted not to exceed 0.5% at micromolar concentrations of protein under equilibrium and both strongly unfolding and refolding conditions for metal-free pseudo-wild-type SOD.

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli.

The role of domains in defining the equilibrium and kinetic folding properties of dihydrofolate reductase (DHFR) from Escherichia coli was probed by examining the thermodynamic and kinetic properties of a set of variants in which the chain connectivity in the discontinuous loop domain (DLD) and the adenosine-binding domain (ABD) was altered by permutation. To test the concept that chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident, permutations were introduced at one position within the ABD, one within the DLD and one at a boundary between the domains. The results demonstrated that a continuous ABD is required for a stable thermal intermediate and a continuous DLD is required for a stable urea intermediate. The permutation at the domain interface had both a thermal and urea intermediate. Strikingly, the observable kinetic folding responses of all three permuted proteins were very similar to the wild-type protein. These results demonstrate a crucial role for stable domains in defining the energy surface for the equilibrium folding reaction of DHFR. If domain connectivity affects the kinetic mechanism, the effects must occur in the sub-millisecond time range.

The coordination of the isomerization of a conserved non-prolyl cis peptide bond with the rate-limiting steps in the folding of dihydrofolate reductase.

The propensity for peptide bonds to adopt the trans configuration in native and unfolded proteins, and the relatively slow rates of cis-trans isomerization reactions, imply that the formation of cis peptide bonds in native conformations are likely to limit folding reactions. The role of the conserved cis Gly95-Gly96 peptide bond in dihydrofolate reductase (DHFR) from Escherichia coli was examined by replacing Gly95 with alanine. The introduction of a beta carbon at position 95 is expected to increase the propensity for the trans isomer and perturb the isomerization reaction required to reach the native conformation. Although G95A DHFR is 1.30 kcal mol(-1) less stable than the wild-type protein, it adopts a well-folded structure that can be chemically denatured in a cooperative fashion. The mutant protein also retains the complex refolding kinetic pattern attributed to a parallel-channel mechanism in wild-type DHFR. The spectroscopic response upon refolding monitored by Trp fluorescence and the absence of a Trp/Trp exciton coupling apparent in the far-UV CD spectrum of the wild-type protein, however, indicated that the tertiary structure of the folded state for G95A DHFR is altered. The addition of methotrexate (MTX), a tight-binding inhibitor, to folded G95A DHFR restored the exciton coupling and the fluorescence properties through five slow kinetic events whose relaxation times are independent of the ligand and the denaturant concentrations. The results were interpreted to mean that MTX-binding drives the formation of the cis isomer of the peptide bond between Ala95 and Gly96 in five compact and stable but not wild-type-like conformations that contain the trans isomer. Folding studies in the presence of MTX for both wild-type and G95A DHFR support the notion that the cis peptide bond between Gly95 and Gly96 in the wild-type protein forms during four parallel rate-limiting steps, which are primarily controlled by folding reactions, and lead directly to a set of native, or native-like, conformers. The isomerization of the cis peptide bond is not a source of the parallel channels that characterize the complex folding mechanism for DHFR.