No abstract

Amyotrophic lateral sclerosis, or Lou Gehrig's disease, is characterized by motor neuron death, with average survival times of two to five years. One cause of this disease is the misfolding of superoxide dismutase 1 (SOD1), a phenomenon influenced by point mutations spanning the protein. Herein, we used an epitope-specific high-throughput screen to identify a peptide ligand that stabilizes the SOD1 native conformation and accelerates its folding by a factor of 2.5. This strategy may be useful for fundamental studies of protein energy landscapes as well as designing new classes of therapeutics.

Amyotrophic lateral sclerosis,o rL ou Gehrigs disease,i sc haracterized by motor neuron death, with average survival times of two to five years.One cause of this disease is the misfolding of superoxidedismutase 1( SOD1), aphenomenon influenced by point mutations spanning the protein.Herein, we used an epitope-specific high-throughput screen to identify ap eptide ligand that stabilizes the SOD1 native conformation and accelerates its folding by afactor of 2.5. This strategy mayb eu seful for fundamental studies of protein energy landscapes as well as designing new classes of therapeutics. Figure 5. Folding rate constantsfor apo WT SOD1, combinedi nto achevron plot, reveal an enhancement in the folding rate in the presence of ligand by afactor of 2.48, without changing the ratelimiting transition structure (p < 0.005).

Protein catalyzed capture agents are an emerging class of oligopeptides that combine the benefits of small molecules and antibodies to furnish ligands with picomolar binding affinity, serum stability, and cell permeability. Their identification involves screening a synthetic, alkyne-functionalized epitope from a target protein against a library of cyclic peptides bearing terminal azides. We identified ligands that bind regions of superoxide dismutase 1 (SOD1), a protein that misfolds to cause amyotrophic lateral sclerosis (ALS), consistently destabilized upon mutation. Treatment of the disease is challenging because there are over 180 heritable mutations of SOD1 and virtually no well-defined binding sites addressable by traditional ligand identification strategies. These mutations ultimately cause the protein to adopt toxic conformations that aggregate and damage cellular functions within the central nervous system. PCC agents targeting regions consistently destabilized across several mutations bind and stabilize its native conformation. We characterized the impact of binding, both on the ground state stability of several mutants as well as the kinetics of SOD1 folding and denaturation.

aggregation phenomena. Amorphous aggregation of the g-crystallins in the eye lens causes a widespread disease of aging, cataract. We combined simulations and experiments to study the mechanism of aggregation of two gD-crystallin mutants, the congenital cataract mutation W42R and the mimic of agerelated oxidative damage W42Q. The two mutants had highly similar properties. We found that formation of an internal disulfide bond was necessary and sufficient for aggregation under physiological conditions in vitro. Twochain all-atom Monte Carlo simulations predicted that one non-native disulfide in particular, between Cys32 and Cys41, was likely to stabilize an unfolding intermediate prone to specific intermolecular interactions. Mass spectrometry detected this internal bond in aggregates formed under physiological conditions in vitro; mutagenesis experiments confirmed that it is needed for aggregation; its formation is also consistent with a recent in vivo proteomic study. Mining our simulation results linked formation of this disulfide to specific conformational changes: extrusion of the N-terminal b-hairpin and shortening of the linker between it and the domain core. Specific binding between the extruded hairpin and a distal b-sheet in an intermolecular chain reaction similar to domain swapping is the most probable mechanism of aggregate propagation.