Folding and aggregation are selectively influenced by the conformational preferences of the alpha-helices of muscle acylphosphatase

The native state of human muscle acylphosphatase (AcP) presents two alpha - helices. In this study we have investigated folding and aggregation of a nu mber of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding i ndicate that only helix-2, spanning residues 55-67, is largely stabilized i n the transition state for folding therefore playing a relevant role in thi s process. On the contrary, the aggregation rate appears to vary only for t he variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first he lix slow down the aggregation process while those that destabilize it incre ase the aggregation rate. AcP variants with the first helix destabilized ag gregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta -sheet structur e. The fact that the first a-helix is important for aggregation and the sec ond helix is important for folding indicates that these processes are highl y specific. This partitioning does not reflect the difference in intrinsic alpha -helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregatio n do not therefore initiate from regions that have simply secondary structu re propensities favorable for such processes. The identification of the reg ions involved in aggregation and the understanding of the factors that prom ote such a process are of fundamental importance to elucidate the principle s by which proteins have evolved and for successful protein design.