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

Significance β2-Microglobulin is an abundant and normally soluble protein. In patients undergoing chronic dialysis, however, it forms insoluble amyloid plaques, leading to medical complications. It has been suggested that the conformational transformation of soluble protein monomers into polymeric amyloids is mediated by isomerization of a single amino acid, namely, proline 32. In this study, we probed the role of this amino acid by chemically synthesizing uniquely tailored protein analogs containing noncanonical amino acids at position 32. Our results show that both the chemical equilibrium and rate of cis-trans isomerization of proline 32 are critical for the solubility of β2-microglobulin and its self-assembly into morphologically distinct amyloid fibrils. These insights may aid ongoing efforts to provide remedies against dialysis-related amyloidosis. 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 cis-trans 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 composition—incorporation 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.

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