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

Minkoo Ahn,Anne Dhulesia,Ana Bernardo-Gancedo,Christopher M Dobson,Erwin De Genst,Janet R Kumita,Christopher A Waudby,Xavier Salvatella,John Christodoulou

doi:10.1038/s41598-017-14739-5

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.

Highlights

To investigate the intermolecular interactions between transiently populated human lysozyme molecules by paramagnetic relaxation enhancement (PRE), we first need to incorporate appropriate probes
With the exception of metalloproteins that contain intrinsic paramagnetic groups, most proteins must be labeled with an extrinsic paramagnetic group in order to conduct PRE measurements and a range of spin labeling techniques have been applied to PRE measurements of proteins[12,17]
Central to specific spin labeling of protein molecules for PRE measurement by NMR has been the use of site-directed mutagenesis to substitute a desired amino acid with a single cysteine residue, which can subsequently be reacted with thiol-reactive reagents, such as MTSL18

Summary

Introduction

To investigate the intermolecular interactions between transiently populated human lysozyme molecules by PREs, we first need to incorporate appropriate probes. In this study we have aimed to incorporate nitroxide spin labels using our selective lysine labeling strategy[25] in order to investigate transient intermolecular interactions between monomeric species present during the early events of lysozyme fibril formation. Intramolecular PRE experiments of spin-labeled I59T lysozyme variants, under native conditions, were used to confirm the functionality and applicability of PRE NMR experiments (Fig. 1). These spin-labeled proteins were further studied, under fibril forming conditions, and intermolecular PREs between transiently populated species were observed, demonstrating that the earliest interactions occur between the unfolded β-domain region of the non-native species

Methods

Results

Discussion

Conclusion