Glycine at Position 93 in SOD1: Mutation-Sensitivity Landscape and Context-Dependent Folding Requirements

Glycine, the simplest amino acid with a single hydrogen atom as its side chain, plays a crucial role in protein folding and structural flexibility. In this study, we used copper/zinc superoxide dismutase (SOD1) as a model system to investigate how the substitution of glycine at position 93 with various amino acids affects the protein structure and stability. We engineered 19 G93 SOD1 mutants and evaluated their folding patterns and aggregation propensities using immunoblotting, fluorescence microscopy, and fluorescence loss in photobleaching (FLIP) assays. All mutants, regardless of the amino acid substitution, form protein aggregates with varying degrees of stability, demonstrating that position 93 exhibits extreme mutation sensitivity, with different substitutions producing distinct destabilization pathways. Our findings demonstrate that alteration of glycine's minimal side chain─consisting of a single hydrogen atom─ disrupts native protein structure. The physicochemical properties of the substituting amino acid, such as polarity, charge, and steric bulk, critically modulate the nature and extent of the resulting misfolding. Nonpolar residues promote aggregation primarily through hydrophobic interactions, while polar and charged residues drive aggregation via hydrogen bonding and electrostatic interactions. This study provides fundamental insights into glycine's unique structural contributions to protein architecture and presents a conceptual framework for understanding how side chain properties influence protein folding and stability. These findings also provide mechanistic implications for protein aggregation processes in neurodegenerative diseases.