Structural and Dynamics Characteristics of Acylphosphatase from Sulfolobus solfataricus in the Monomeric State and in the Initial Native-like Aggregates

Katiuscia Pagano,Francesco Bemporad,Federico Fogolari,Gennaro Esposito,Paolo Viglino,Fabrizio Chiti,Alessandra Corazza

doi:10.1074/jbc.m109.082156

Abstract

It has previously been shown that the acylphosphatase from Sulfolobus solfataricus is capable of forming amyloid-like aggregates under conditions in which the native structure is maintained and via the transient formation of native-like aggregates. Based on the previously determined NMR structure of the native protein, showing a ferredoxin-like fold and the peculiar presence of an unstructured N-terminal segment, we show here, at a molecular level using NMR spectroscopy, that indeed S. solfataricus acylphosphatase remains in a native-like conformation when placed in aggregating conditions and that such a native-like structure persists when the protein forms the initial aggregates, at least within the low molecular weight species. The analysis carried out under different solution conditions, based on the measurement of the combined (1)H and (15)N chemical shifts and hydrogen/deuterium exchange rates, enabled the most significant conformational changes to be monitored upon transfer of the monomeric state into aggregating conditions and upon formation of the initial native-like aggregates. Important increases of the hydrogen/deuterium exchange rates throughout the native protein, accompanied by small and localized structural changes, in the monomeric protein were observed. The results also allow the identification of the intermolecular interaction regions within the native-like aggregates, that involve, in particular, the N-terminal unstructured segment, the apical region including strands S4 and S5 with the connecting loop, and the opposite active site.

Highlights

Proteins and peptides have a generic propensity to form well organized aggregates characterized by a fibrillar morphology and an extended cross-␤ structure, generally referred to as amyloid-like fibrils [1, 2]
Sso AcP induces an 11-fold increase in ThT (v/v) TFE, Sso AcP does not induce any significant increase in fluorescence only in acetate buffer supplemented with 20% ThT fluorescence, confirming that no amyloid-like aggregates (v/v) TFE (Fig. 1)
Exponential kinetics and was previously shown to be related to Sso AcP Retains a Native-like Structure in the Initial Small amyloid-like aggregation, protofibril formation [8]

Summary

Introduction

Proteins and peptides have a generic propensity to form well organized aggregates characterized by a fibrillar morphology and an extended cross-␤ structure, generally referred to as amyloid-like fibrils [1, 2]. In a first rapid phase, the native protein aggregates within 2 min into oligomers in which the individual molecules still possess considerable enzymatic activity These oligomeric species do not bind Congo red, thioflavin T (ThT), or 1-anilino8-naphtalenesulfonic acid and are not yet characterized by the high content of ␤-sheet structure typical of amyloid aggregates [11]. These initial native-like aggregates convert directly, within 30 min, into spherical and chain-like protofibrils. These latter species have a diameter of 3–5 nm; have no enzymatic activity; bind Congo red, ThT, and 1-anilino-8-naphtalenesulfonic acid; and have a large ␤-sheet structure content [11]. Both phases of aggregation are more rapid than the unfolding reaction determined for the monomeric, native state under the same conditions, indicating that unfolding is not a necessary step in the process

Methods

Results

Conclusion