Amyloid Formation of a Protein in the Absence of Initial Unfolding and Destabilization of the Native State

Abstract

In 5% (v/v) trifluoroethanol, pH 5.5, 25 degrees C one of the acylphosphatases from Drosophila melanogaster (AcPDro2) forms fibrillar aggregates that bind thioflavin T and Congo red and have an extensive beta-sheet structure, as revealed by circular dichroism. Atomic force microscopy indicates that the fibrils and their constituent protofilaments have diameters compatible with those of natural amyloid fibrils. Spectroscopic and biochemical investigation, carried out using near- and far-UV circular dichroism, intrinsic and 1-anilino-8-naphthalenesulfonic acid-derived fluorescence, dynamic light scattering, and enzymatic activity assays, shows that AcPDro2 has, before aggregation, a secondary structure content packing around aromatic and hydrophobic residues, hydrodynamic diameter, and catalytic activity indistinguishable from those of the native protein. The native protein was found to have the same conformational stability under native and aggregating conditions, as determined from urea-induced unfolding. The kinetic analysis supports models in which AcPDro2 aggregates initially without need to unfold and subsequently undergoes a conformational change into amyloid-like structures. Although fully or partially unfolded states have a higher propensity to aggregate, the residual aggregation potential that proteins maintain upon complete folding can be physiologically relevant and be directly involved in the pathogenesis of some protein deposition diseases.

Experimental free energy surfaces reveal the mechanisms of maintenance of protein solubility

“…Moreover, within experimental error, AcPDro2 has the same thermodynamic stability (i.e., the same free energy of unfolding, ΔG U-F ) in the presence and in the absence of 5% (vol∕vol) TFE as determined using urea-induced denaturation at equilibrium (19). The similar thermodynamic stabilities in 0% and 5% (vol∕vol) TFE originate from the fact that the folding and unfolding rates are accelerated to similar extents following the addition of 5% (vol∕vol) TFE (19). By contrast to AcPDro2, most folded proteins aggregate in the presence of much higher concentrations of TFE (15-30%) where a significant portion of the molecules are unfolded or strongly destabilized.…”

“…Indeed, under these conditions, the hydrodynamic radius, intrinsic fluorescence, secondary structure content, and enzymatic activity of AcPDro2 in its monomeric state are indistinguishable with those of the protein in the absence of TFE, where the propensity of AcPDro2 to aggregate is extremely low (19). Moreover, within experimental error, AcPDro2 has the same thermodynamic stability (i.e., the same free energy of unfolding, ΔG U-F ) in the presence and in the absence of 5% (vol∕vol) TFE as determined using urea-induced denaturation at equilibrium (19). The similar thermodynamic stabilities in 0% and 5% (vol∕vol) TFE originate from the fact that the folding and unfolding rates are accelerated to similar extents following the addition of 5% (vol∕vol) TFE (19).…”

“…AcPDro2 in its native state is a globular and monomeric protein with a structure consisting of five β-strands (S1-S5), which form a single β-sheet, and two α-helices (H1 and H2) that lie adjacent to this α-sheet. The importance that subtle intrinsic factors play in enabling this protein to remain soluble is clearly shown by the fact that a very low concentration (5% vol∕vol) of trifluoroethanol (TFE) is sufficient to induce rapid formation of amyloid fibrils, although the protein still populates a highly native-like conformational ensemble before aggregation occurs (19). Indeed, under these conditions, the hydrodynamic radius, intrinsic fluorescence, secondary structure content, and enzymatic activity of AcPDro2 in its monomeric state are indistinguishable with those of the protein in the absence of TFE, where the propensity of AcPDro2 to aggregate is extremely low (19).…”

“…The importance that subtle intrinsic factors play in enabling this protein to remain soluble is clearly shown by the fact that a very low concentration (5% vol∕vol) of trifluoroethanol (TFE) is sufficient to induce rapid formation of amyloid fibrils, although the protein still populates a highly native-like conformational ensemble before aggregation occurs (19). Indeed, under these conditions, the hydrodynamic radius, intrinsic fluorescence, secondary structure content, and enzymatic activity of AcPDro2 in its monomeric state are indistinguishable with those of the protein in the absence of TFE, where the propensity of AcPDro2 to aggregate is extremely low (19). Moreover, within experimental error, AcPDro2 has the same thermodynamic stability (i.e., the same free energy of unfolding, ΔG U-F ) in the presence and in the absence of 5% (vol∕vol) TFE as determined using urea-induced denaturation at equilibrium (19).…”

The Mechanism of the Amyloidogenic Conversion of T7 Endonuclease I

“…If the amyloidogenic conformer did not exist in the protein conformational ensemble, fibrillization would not occur by simply changing the relative energy level of various conformers. Specific conformational changes may include retention of some native structures, as several studies suggest that amyloid formation can start from native-like state (17,(31)(32)(33)(34). In the case of domain-swapped proteins such as T7EI, the dimer interface has to be preserved in order to take advantage of native interactions.…”