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

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