Multidimensional structure-activity relationship of a protein in its aggregated states

Abstract

Protein aggregates are both associated with disease and function. Because a variety of factors induce protein aggregation, a given protein can aggregate into different states. Here, we compare the structures and activities of five distinct protein aggregates of a single protein. Despite the diverse chemical, physical and biological treatments used to induce aggregation, all aggregate types contain the cross-β-sheet motif. However, they are structurally distinct, having different segments of the protein sequence involved in secondary structure formation. Because of these structural differences each aggregate has a unique set of properties. These include affinity to ATP, Thioflavin T, DNA, and membrane mimics, and interference with cell viability. The key to their multiple properties may be that the repetitive nature of the cross-β-sheet motif guarantees for many potent activities through cooperativity. The observed multidimensional structure-activity relationship of protein aggregates may be important for amyloid diseases but may also be advantageous in nanotechnology.

Figure 1

(a) Electron micrographs (EM) (left) and X-ray fiber diffraction patterns (right) of the five aggregate types of HypF-N as indicated. Scale bars indicate 500 nm. Fibrils have a width of 6±2 nm, inclusion bodies have an irregular sphere-like shape with a radius of 280±40 nm, heat-precipitated aggregates also have an irregular sphere-like shape with a radius of 80±20 nm, concentration-induced and TCA-precipitated aggregates have amorphous morphology with widely ranging sizes, with the latter having irregular sphere-like substructures with a radius of 20±4nm. (±: standard error). The sharp reflection of the fiber diffraction pattern observed at approximately 4.7 Å is interpreted as the spacing between strands in a β-sheet, and the diffuse reflection at approximately 10 Å is interpreted as the spacing between β-sheets. (b) FT-IR spectra of HypF-N aggregates and soluble HypF-N. Fibrils (green); inclusion bodies (cyan); heat aggregates (blue), concentration aggregates (yellow); TCA aggregates (red); soluble HypF-N (purple). In the Amide I region, soluble HypF-N shows two major peaks around 1633 cm⁻¹ and 1657 cm⁻¹ that are usually assigned to intramolecular β-sheet and α-helix secondary structures.[21] For fibrils, there is a sharp peak around 1624 cm⁻¹ and a peak around 1694 cm⁻¹ that are indicative of newly formed β-sheet structure in the aggregate.[21] While for TCA-precipitated aggregates only little newly formed β-sheet structure is observed, the other three aggregate entities contain increased amounts of β-sheet structure. Spectra were normalized at the tyrosine band around 1513 cm⁻¹ to account for differences in the total protein content.

Figure 2

Sequence-resolved amide exchange rates k_ex/h indicative of secondary structures for all five types of HypF-N aggregates studied as indicated. The measured H/D exchange is mostly of bi-exponential nature suggesting the presence of two populations. The exchange rates of the major population are colored green. If the minor population is present more than 1/3, the corresponding exchange rates are shown in grey. Because of spectral overlap, the analysis of the exchange rates for some residues may be difficult to determine. However, most of these overlap problems could be resolved by the assumption that sequential neighboring residues show a similar extent of exchange. The exchange rates that have been extracted following this procedure are colored in light green. In the panel labeled with “A”, predicted aggregation-prone segments of HypF-N are shown using two distinct algorithms: 3DPROFILE in orange and TANGO in blue. The secondary structures of the soluble HypF-N are highlighted in red for α-helix and blue for β-sheet, respectively. For details of prediction algorithms, please see Supporting Information.

Figure 3

Activities of HypF-N aggregates. (a) Urea disaggregation of HypF-N aggregates and unfolding of soluble HypF-N as labeled. (b) ATP binding capability of different aggregates. ATP and aggregates are mixed at a 1:5 monomer ratio, and the column values represent the percentage of bound ATP vs. total amount of ATP. (c) ThT fluorescence enhancement upon binding to different aggregates. The column values represent the fluorescence intensity at arbitrary unit (AU). (d) DNA binding capability of different aggregates. DNA and aggregates are mixed at a 1:20 monomer ratio, and the column values represent the percentage of bound DNA vs. total amount of DNA. (e) DHPC micelle binding capability of different aggregates. DHPC micelle and aggregates are mixed at a 1:1 concentration ratio and the column values represent the percentage of bound DHPC micelles vs. total amount of DHPC micelles. (f) MTT cell reduction upon addition of different aggregates. The column values represent the percentage of reduced cells with addition of aggregates vs. reduced cells without aggregates. Aβ_1-42 amyloid fibrils were measured as a positive control. Activities of soluble HypF-N were also measured as reference. It should be noted that HypF-N fibrils do not show an influence in MTT reduction on the human neurotypic SH-SY5Y cells line used by ref [35] when sent by us to the Chiti group. At least two independent experiments were carried out for each system. (±: standard error of the experimental measurements for individual preparations)