Fluoroalcohol-Induced Modulation of the Pathway of Amyloid Protofibril Formation by Barstar

Abstract

To understand how the conformational heterogeneity of protofibrils formed by any protein, as well as the mechanisms of their formation, are modulated by a change in aggregation conditions, we studied the formation of amyloid protofibrils by barstar at low pH by multiple structural probes in the presence of hexafluoroisopropanol (HFIP). In the presence of 10% HFIP, aggregation proceeds with the transient formation of spherical oligomers and leads to the formation of both protofibrils and fibrils. Curly short protofibrils and fibrils are seen to form early during the aggregation reaction, and both are seen to grow gradually in length during the course of the reaction. Atomic force microscopy images reveal that the HFIP-induced protofibrils are long (∼300 nm in length), curly, and beaded and appear to be composed primarily of β-sheet bilayers, with heights of ∼2.4 nm. The protofibrils formed in the presence of HFIP differ in both their structures and their stabilities from the protofibrils formed either in the absence of alcohol or in the presence of a related alcohol, trifluoroethanol (TFE). Aggregation appears to proceed via an isodesmic polymerization mechanism. Internal structure in the growing aggregates changes in two stages during protofibril formation. In the first stage, an α-helix-rich oligomeric intermediate is formed. In the second stage, the level of β-sheet structure increases at the expense of some α-helical structure. The second stage itself appears to occur in two distinct steps. The creation of thioflavin T binding sites occurs concomitantly with aggregate elongation and is seen to precede the change in secondary structure. The long straight fibrils with characteristic heights of 8-10 nm, which form in the course of the HFIP-induced aggregation reaction, have not been observed to form either in the absence of alcohol or in the presence of TFE.

Solvent-Induced Tuning of Internal Structure in a Protein Amyloid Protofibril

“…NMR studies have shown that the core of the A form is formed by the last two-thirds of the primary structure (34). The A form can be transformed into protofibrils in various aggregation conditions (21,25,32,35). Fluorescence measurements have shown that the residue-specific pattern of side-chain dynamics in the A form is similar to that in protofibrils, a result consistent with the A form being the direct precursor of the protofibrils (33).…”

“…Fluorescence measurements have shown that the residue-specific pattern of side-chain dynamics in the A form is similar to that in protofibrils, a result consistent with the A form being the direct precursor of the protofibrils (33). Atomic force microscopy (AFM) (21,25,33) and transmission electron microscopy (21) studies have shown that barstar protofibrils are curvilinear fibrillar structures several hundred nanometers in length. Interestingly, protofibrils formed by barstar under different aggregation conditions differ in both external appearance and size, as well as in stability (21,25).…”

“…High-resolution structural characterization of the conformational diversity in amyloidlike aggregates has been restricted so far to the mature aggregates known as amyloid fibrils (18)(19)(20). It has been more difficult to carry out highresolution structural characterization of the curvilinear amyloid protofibrils and spherical oligomers that are seen to form during the course of amyloid fibril formation (1,(21)(22)(23), or for the wormlike fibrils seen to be formed by the prion protein, as well as by b-microglobulin (8). Although conformational heterogeneity in such aggregates has been identified (24)(25)(26), there is little understanding of how such heterogeneity is related to the primary structure of the protein.…”

Slow Amyloid Nucleation via α-Helix-Rich Oligomeric Intermediates in Short Polyglutamine-Containing Huntingtin Fragments

“…Similar mechanisms have been suggested for other amyloid systems, 55 including α-synuclein, 56 amyloid β, 57 islet amyloid polypeptide, 58 and several model systems. 59,60 The htt system, however, appears to be unique in that a substantial portion of the α-helix that is the basis of oligomer formation is actually retained in the final fibrils 21 (Fig. 8b).…”

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

“…Protein polymerization mechanisms are broadly classified under two prevalent models, namely the nucleation-elongation polymerization model and the isodesmic polymerization model [3], [4]. The latter however, is rarely encountered with few reports in protein literature till date [5], [6]. The absence of a critical concentration in isodesmic polymerization implies that aggregation can occur at nanomolar monomer concentrations which can be exploited to investigate molecular features of aggregates.…”