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.