Direct Observation of Defects and Increased Ion Permeability of a Membrane Induced by Structurally Disordered Cu/Zn-Superoxide Dismutase Aggregates

Interactions between protein aggregates and a cellular membrane have been strongly implicated in many protein conformational diseases. However, such interactions for the case of Cu/Zn superoxide dismutase (SOD1) protein, which is related to fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS), have not been explored yet. For the first time, we report the direct observation of defect formation and increased ion permeability of a membrane induced by SOD1 aggregates using a supported lipid bilayer and membrane patches of human embryonic kidney cells as model membranes. We observed that aggregated SOD1 significantly induced the formation of defects within lipid membranes and caused the perturbation of membrane permeability, based on surface plasmon resonance spectroscopy, atomic force microscopy and electrophysiology. In the case of apo SOD1 with an unfolded structure, we found that it bound to the lipid membrane surface and slightly perturbed membrane permeability, compared to other folded proteins (holo SOD1 and bovine serum albumin). The changes in membrane integrity and permeability were found to be strongly dependent on the type of proteins and the amount of aggregates present. We expect that the findings presented herein will advance our understanding of the pathway by which structurally disordered SOD1 aggregates exert toxicity in vivo

Performance Analysis of Orthogonal Pairs Designed for an Expanded Eukaryotic Genetic Code

Background: The suppression of amber stop codons with non-canonical amino acids (ncAAs) is used for the site-specific introduction of many unusual functions into proteins. Specific orthogonal aminoacyl-tRNA synthetase (o-aaRS)/amber suppressor tRNA CUA pairs (o-pairs) for the incorporation of ncAAs in S. cerevisiae were previously selected from an E. coli tyrosyl-tRNA synthetase/tRNACUA mutant library. Incorporation fidelity relies on the specificity of the o-aaRSs for their ncAAs and the ability to effectively discriminate against their natural substrate Tyr or any other canonical amino acid. Methodology/Principal Findings: We used o-pairs previously developed for ncAAs carrying reactive alkyne-, azido-, or photocrosslinker side chains to suppress an amber mutant of human superoxide dismutase 1 in S. cerevisiae. We found worse incorporation efficiencies of the alkyne- and the photocrosslinker ncAAs than reported earlier. In our hands, amber suppression with the ncAA containing the azido group did not occur at all. In addition to the incorporation experiments in S. cerevisiae, we analyzed the catalytic properties of the o-aaRSs in vitro. Surprisingly, all o-aaRSs showed much higher preference for their natural substrate Tyr than for any of the tested ncAAs. While it is unclear why efficiently recognized Tyr is not inserted at amber codons, we speculate that metabolically inert ncAAs accumulate in the cell, and for this reason they are incorporated despite being weak substrates for the o-aaRSs. Conclusions/Significance: O-pairs have been developed for a whole plethora of ncAAs. However, a systematic and detaile