<p></p><p>Protein-protein interactions
(PPIs) are involved in many of life’s essential biological functions yet are
also an underlying cause of several human diseases, including amyloidosis. The
modulation of PPIs presents opportunities to gain mechanistic insights into
amyloid assembly, particularly through the use of methods which can trap
specific intermediates for detailed study. Such information can also provide a
starting point for drug discovery. Here, we demonstrate that covalently
tethered small molecule fragments can be used to stabilize specific oligomers
during amyloid fibril formation, facilitating the structural characterization
of these assembly intermediates. We exemplify the power of covalent tethering
using the naturally occurring truncated variant (ΔN6) of the human protein
β2-microglobulin (β2m), which assembles into amyloid fibrils associated with
dialysis-related amyloidosis. Using this approach, we have trapped tetramers
formed by ΔN6 under conditions which would normally lead to fibril formation
and found that the degree of tetramer stabilization depends on the site of the
covalent tether and the nature of the protein-fragment interaction. The
covalent protein-ligand linkage enabled structural characterization of these
trapped oligomeric species using X-ray crystallography and NMR, providing
insight into why tetramer stabilization inhibits amyloid assembly. Our findings
highlight the power of “post-translational chemical modification" as a
tool to study biological molecular mechanisms. </p><br><p></p>
Structural Characterization of the Hydrophobin SC3, as a Monomer and after Self-Assembly at Hydrophobic/Hydrophilic Interfaces
