Edge strand engineering prevents native‐like aggregation in S ulfolobus solfataricus acylphosphatase

β‐proteins are constantly threatened by the risk of aggregation because β‐sheets are inherently structured for edge‐to‐edge interactions. To avoid native‐like aggregation, evolution has resulted in a set of strategies that prevent intermolecular β‐interactions. Acylphosphatase from S ulfolobus solfataricus ( S so  A c P ) represents a suitable model for the study of such a process. Under conditions promoting aggregation, S so  A c P acquires a native‐like conformational state whereby an unstructured N ‐terminal segment interacts with the edge β‐strand B 4 of an adjacent S so  A c P molecule. Because B 4 is poorly protected against aggregation, this interaction triggers the aggregation cascade without the need for unfolding. Recently, three single S so  A c P mutants ( V 84 D , Y 86 E and V 84 P ) were designed to engineer additional protection against aggregation in B 4 and were observed to successfully impair native‐like aggregation in all three variants at the expense of a lower stability. To understand the structural basis of the reduced aggregation propensity and lower stability, the crystal structures of the S so  A c P variants were determined in the present study. Structural analysis reveals that the V 84 D and Y 86 E mutations exert protection by the insertion of an edge negative charge. A conformationally less regular B 4 underlies protection against aggregation in the V 84 P mutant. The thermodynamic basis of instability is discussed. Moreover, kinetic experiments indicate that aggregation of the three mutants is not native‐like and is independent of the interaction between B 4 and the unstructured N ‐terminal segment. The reported data rationalize previous evidence regarding S so  A c P native‐like aggregation and provide a basis for the design of aggregation‐free proteins. Database The atomic coordinates and related experimental data for the S so  A c P mutants V 84 P , V 84 D , Δ N 11 Y 86 E have been deposited in the Protein Data Bank under accession numbers 4OJ3 , 4OJG and 4OJH , respectively. Structured digital abstract • Sso AcP and Sso AcP bind by fluorescence technology ( View interaction ).