Application of Lysine-specific Labeling to Detect Transient Interactions Present During Human Lysozyme Amyloid Fibril Formation

Abstract

Populating transient and partially unfolded species is a crucial step in the formation and accumulation of amyloid fibrils formed from pathogenic variants of human lysozyme linked with a rare but fatal hereditary systemic amyloidosis. The partially unfolded species possess an unstructured β-domain and C-helix with the rest of the α-domain remaining native-like. Here we use paramagnetic relaxation enhancement (PRE) measured by NMR spectroscopy to study the transient intermolecular interactions between such intermediate species. Nitroxide spin labels, introduced specifically at three individual lysine residues, generate distinct PRE profiles, indicating the presence of intermolecular interactions between residues within the unfolded β-domain. This study describes the applicability to PRE NMR measurements of selective lysine labeling, at different sites within a protein, as an alternative to the introduction of spin labels via engineered cysteine residues. These results reveal the importance of the β-sheet region of lysozyme for initiating self-assembly into amyloid fibrils.

Serum amyloid P component promotes formation of distinct aggregated lysozyme morphologies and reduces toxicity in Drosophila flies expressing F57I lysozyme

“…We found that SAP can counteract the toxicity induced by the expression of F57I by promoting the formation of alternative morphologies of aggregated lysozyme with more amyloidogenic characteristics. We tested the effect of SAP on in vitro lysozyme fibril formation, using the well-studied I59T lysozyme system [21,22] and confirmed that the presence of SAP promoted enhanced ThT Competing interests: The authors have declared that no competing interests exist.…”

Man does not live by intrinsically unstructured proteins alone: The role of structured regions in aggregation

“…In these intermediates, the β-domain is largely unfolded and able to establish inter-molecular contacts that drive the aggregation of the protein. [59] The aggregation rate of human lysozyme largely correlates with the destabilisation of the native state of the protein and the formation of intermediates. [60,61] In vivo, human lysozyme misfolding occurs as a consequence of pathological mutations.…”

Enhancing Protein Crystal Nucleation Using In Situ Templating on Bioconjugate-Functionalized Nanoparticles and Machine Learning

“…We chose to study lysozyme and insulin, two clinically relevant, well-characterized proteins which crystallize readily and whose self-assembly kinetics have been explored as a function of protein structure. 14 In this study, we evaluate the impact of these bioconjugates on protein crystallization and examine how they affect protein crystallization kinetics.…”

“…Indeed, proteins naturally self-assemble on the nano- and microscale to form highly ordered structures such as crystals, fibrils, or amorphous aggregates . Previous work has shown that intermolecular interactions drive protein aggregation and fibrillation. , In addition, nanofibril structures can even be designed by modifying specific bonds or amino acid residues . A surface of highly ordered proteins can drive the protein crystal nucleation in the same way that seeding a solution with small protein crystals (often practiced in large-scale crystallization) can enhance protein crystal growth.…”

“…Both react with specific amino acids (shown in Figure b)MAL reacts with the thiol group present in cysteine and NHS reacts with the primary amine in lysine or the N-terminus of proteins. We chose to study lysozyme and insulin, two clinically relevant, well-characterized proteins which crystallize readily and whose self-assembly kinetics have been explored as a function of protein structure . In this study, we evaluate the impact of these bioconjugates on protein crystallization and examine how they affect protein crystallization kinetics.…”