title: 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 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.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Vladimir Yu
authors.initials: VY
authors.lastname: Torbeev
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D000577:Amides / Q000737:chemistry*; D000686:Amyloidosis / Q000209:etiology / Q000517:prevention & control*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003956:Dialysis / Q000009:adverse effects*; D006801:Humans; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D012995:Solubility; D001613:beta 2-Microglobulin / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D000577:Amides; D001613:beta 2-Microglobulin; D011392:Proline
keywords: amyloidogenesis; native chemical ligation; polymorphism; protein conformation