Both the cis - trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

Abstract

The human protein β2-microglobulin (β2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger β2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that β2m's stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cistrans isomerization. As anticipated, the β2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical compositionincorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein-modulated the aggregation properties of β2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-β structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in β2m aggregation.protein conformation | polymorphism | amyloidogenesis | native chemical ligation

4-Fluoroprolines: Conformational Analysis and Effects on the Stability and Folding of Peptides and Proteins

“…The first total synthesis of a 4-fluoroproline protein was achieved using β2-microglobulin (β2m), the causative agent of dialysis-related amyloidosis [121]. In the native structure of β2m, the amide of one of the five proline residues, Pro32, adopts a cis isomer [122], which isomerizes to the trans conformation in the amyloid, as shown by solid-state NMR spectroscopy [123].…”

On the Split Personality of Penultimate Proline

“…Cis - trans isomerization of peptide bonds is one mechanism that can lead to large changes in the overall structure of peptides and proteins [5–8]. Such isomers are most commonly associated with proline, primarily because of the 20 natural amino acids proline is unique in that it has a secondary amine at the backbone nitrogen atom [9, 10].…”

C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice

“…One plausible explanation for this hypothesis could be the ability of N62 to engage in an amide zipper to stabilize an intermolecular β-sheet in which it is found. Other reports suggest that the amide in the amyloidogenic regions of proteins such as β 2 M and amylin are associated with amyloidogenicity (37,40,41). However, the 3D structure of mouse apoA-II is unknown, and its role in polymerization is unclear.…”