Folding and Aggregation Are Selectively Influenced by the Conformational Preferences of the α-Helices of Muscle Acylphosphatase

Abstract

The native state of human muscle acylphosphatase (AcP) presents two ␣-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form ␤-sheet structure. The fact that the first ␣-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic ␣-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.

Amyloid-like Fibril Formation in an All β-Barrel Protein

“…Formation of non-native extended ␤-sheet conformation appears to promote aggregation, and induction of non-native helix conformation correlates with the inhibition of protein aggregation. Similar observations were made by Dobson and co-workers, based on exhaustive point mutations in the muscle acylphosphatase protein (35,36). A clear-cut kinetic partitioning between aggregation and folding of proteins was observed.…”

Modulating Calcitonin Fibrillogenesis

“…LAsCT showed properties identical to those of sCT, the only difference being the stability and the length of the amphipathic ␣-helix in membrane-like environment. 2 We report here that, in water, LAsCT forms an ␣-helical head-to-tail dimer in the region Leu 12 -Tyr 22 . Guanidine HCl unfolding experiments at physiological pH indicate that hydrophobic interactions are responsible for the association of all three calcitonins.…”

“…It is not known which structural features cause specific proteins to form amyloid fibrils in vivo; however, evidence is accumulating that aggregation is initiated from specific regions within a polypeptide chain (2)(3)(4)(5)(6).…”

The intrachain disulfide bridge is responsible of the unusual stability properties of novel acylphosphatase from Escherichia coli

“…Hu-CT [23] 16.6 ± 1.0 a 6.3 ± 0.6 a 2.6 ± 0.2 a 25 Hu-MT [2] 21.3 ± 2.0 a 5.3 ± 0.5 a 4.0 ± 0.4 a 28 AcPDro [7] 10.3 ± 1.0 a 6.5 ± 0.6 a 1.6 ± 0.1 a 25 AcPDro2 [8] 13.9 ± 1.0 a 5.5 ± 0.5 a 2.5 ± 0.2 a 28 SsoAcP [24] 49 Determined with Gdn-HCl. Fig.…”