Covalent Tethering and Residues with Bulky Hydrophobic Side Chains Enable Self‐Assembly of Distinct Amyloid Structures

Abstract

Polymorphism is a common property of amyloid fibers that complicates their detailed structural and functional studies. Here we report experiments illustrating the chemical principles that enable the formation of amyloid polymorphs with distinct stoichiometric composition. Using appropriate covalent tethering we programmed self-assembly of a model peptide corresponding to the [20-41] fragment of human β2-microglobulin into fibers with either trimeric or dimeric amyloid cores. Using a set of biophysical and biochemical methods we demonstrated their distinct structural, morphological, and templating properties. Furthermore, we showed that supramolecular approaches in which the peptide is modified with bulky substituents can also be applied to modulate the formation of different fiber polymorphs. Such strategies, when applied to disease-related peptides and proteins, will greatly help in the evaluation of the biological properties of structurally distinct amyloids.

Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules

“…Covalent small molecules are becoming increasingly attractive tools to modulate specific protein-protein interactions 43,44,[58][59][60] , to interrogate the role of different proteins in biological processes 44,58,[61][62][63] , and to facilitate structural studies of challenging targets 42,64 . The results presented here expand this covalent chemical tool approach, highlighting its power to facilitate analysis of the structure and role of specific oligomers in self-assembly pathways.…”

Visualizing and trapping transient oligomers in amyloid assembly pathways

“…Covalent ligands can offer improved affinity and selectivity over their non-covalent analogues [ 213 , 216 , 217 ], thereby overcoming some of the difficulties associated with using small molecules to target the dynamic and poorly structured oligomers formed by many amyloid proteins. Modification of a protein's behavior through functionalization does not require the conjugated chemical moiety to have a high non-covalent affinity for the target; it can instead effectively act as a chemical post-translational modification [ 218 , 219 ], altering the protein's surface properties [ 27 , 220 ] or acting as a steric block [ 30 ]. We also note that sequence variation can be considered as a special case of protein functionalization which alters the chemistry and/or steric bulk of protein sidechains without the need for chemical modifications to be performed post-translationally.…”

Total chemical synthesis and biophysical properties of a designed soluble 24 kDa amyloid analogue

“…Previously, we demonstrated that [20–41]β2m covalently tethered into a “covalent trimer” aggregates into morphologically distinct amyloids. 10 In this study, we engineered a covalently tethered oligomer composed of three units of the [20–41]β2m covalent trimer. As a result, the final construct comprises nine copies of the progenitor [20–41]β2m peptide in total ( Fig.…”

“…The design of this complex peptide architecture was realized with the help of molecular modelling taking into account the structural properties of the [20–41]β2m covalent trimer. 10 We envisaged three copies of covalent trimer [20–41]β2m stacked in a parallel, in-register orientation, so that the internal covalent trimer being “sandwiched” by two external trimers has surroundings identical to those found in a complete amyloid fiber ( Fig. 1 ).…”

“…In our previous work on covalent trimer [20–41]β2m amyloids, the trivalent linker N , N ′, N ′′-(benzene-1,3,5-triyl)-tris(acetamide) was used to join three copies of the [20–41]β2m peptide. 10 The conjugation was based on a nucleophilic displacement of a symmetrically substituted bromoacetamide derivative by a Cys6 residue of the [20–41]β2m. In this work, according to our design objectives, the central linker needs to support the attachment of different peptides that would enable introduction of the additional C–N linkers.…”