High‐Resolution NMR H/D Exchange of Human Superoxide Dismutase Inclusion Bodies Reveals Significant Native Features Despite Structural Heterogeneity

Abstract

Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

Methodological advances and strategies for high resolution structure determination of cellular protein aggregates

“…Furthermore, it has been shown that bacterial IBs are composed of functional native and native-like protein structures along with amyloid or aggregated forms of the same protein. This molecular architecture allows for retention of biological activity as well as high mechanical stability, showing promise for their use as functional materials ( 8 , 33 ). Consequently, IBs are being explored for applications as drug delivery systems and self-immobilized catalysts.…”

“…Many cell-derived aggregates, including bacterial IBs, show increased β character ( 68 , 69 ), and in some cases, amyloid-like features have been identified in cell-derived IBs using FTIR ( 68 , 70 ). More recently the coexistence of fibril-like intermolecular β-sheets with significant native structure ( 19 , 33 , 71 , 72 ) has further elucidated the heterogeneity of IBs. As the amount of residual native structure in IBs often correlates with the yield of enzymatically active protein, FTIR can be an essential screening tool for researchers trying to understand and optimize functional IBs ( 72 , 73 , 74 ).…”

“…For some time, qHDX was only used sparingly, predominantly for purified protein aggregates ( 125 , 126 ), until a recent publication on SOD1 cellular IB structure by Naser et al. ( 33 ). This study differed from previous work in key aspects.…”

“…Interpretation is often aided by comparison to FTIR spectra of purified proteins. Data are for SOD1, where panel (F) is modified from Naser et al (33).…”

“…CD has high relative absorbance for α-helical structure, while FTIR provides a more uniform signal for different secondary structures allowing for easier visual assessment of structural changes ( 28 , 29 ). At the atomic level, methods such as quenched hydrogen deuterium exchange (qHDX) and solid-state NMR (ssNMR) provide high resolution information on which residues are participating in the aggregate structure, allowing for a mechanistic interpretation of aggregate formation ( 30 , 31 , 32 , 33 , 34 , 35 ). These specific methods will be emphasized later in this review as they provide a wealth of complementary and high resolution information.…”