Nanoimaging for protein misfolding and related diseases

Abstract

Misfolding and aggregation of proteins is a common thread linking a number of important human health problems. The misfolded and aggregated proteins are inducers of cellular stress and activators of immunity in neurodegenerative diseases. They might possess clear cytotoxic properties, being responsible for the dysfunction and loss of cells in the affected organs. Despite the crucial importance of protein misfolding and abnormal interactions, very little is currently known about the molecular mechanism underlying these processes. Factors that lead to protein misfolding and aggregation in vitro are poorly understood, not to mention the complexities involved in the formation of protein nanoparticles with different morphologies (e.g., the nanopores) in vivo. A better understanding of the molecular mechanisms of misfolding and aggregation might facilitate development of the rational approaches to prevent pathologies mediated by protein misfolding. The conventional tools currently available to researchers can only provide an averaged picture of a living system, whereas much of the subtle or short-lived information is lost. We believe that the existing and emerging nanotools might help solving these problems by opening the entirely novel pathways for the development of early diagnostic and therapeutic approaches. This article summarizes recent advances of the nanoscience in detection and characterization of misfolded protein conformations. Based on these findings, we outline our view on the nanoscience development towards identification intracellular nanomachines and/or multicomponent complexes critically involved in protein misfolding.

Figure 1

Scheme of protein misfolding and aggregation. In the scheme normally folded protein can undergo conformational transitions into various misfolded states that may aggregate in different morphologies. AFM images of Aβ peptide aggregated into protofilaments, toroids (rings) and fibrils are shown in plates (A), (B) and (C) respectively.

Figure 2

Folded protein (A) adopts an unfolded state (B) that is the transient state for the misfolded conformation (C).

Figure 3

Scheme explaining the nanoprobing approach for the detection and analysis of misfolded states of the protein. Proteins anchored to the substrate surface and the AFM tip (A) are brought into the contact (B) and then pulled apart (C). The forces holding the complex depend on the protein conformation. They are low for normally folded proteins (the force curve “d”). Large rupture force (e) corresponds to a misfolded sate of the protein (D).

Figure 4

The dependence of the rupture forces on pH for lysozyme. See paper [McAllister et al., 2005] for details.

Figure 5

AFM images of fibrils formed by lysozyme grown at pH2.0 (A), pH2.7 (B) and pH3.7 (C).

Figure 6

The data for pulling Aβ 1-40 peptides N-terminated with cysteine and immobilized on the maleimide-silatrane functionalized mica surfaces and the AFM tips. The force curve is approximated by the WLC (thin black curve). The insert shows the histogram generated from a series of the force curves. See paper [Kransnoslobodtsev, 2005] for specifics.

Figure 7

Ribbon-tube representation of the three-dimensional structural model of the β-barrel transmembrane protein consisting of 10 antiparallel β-strands formed by the five adjacent Aβ peptides.

Figure 8

AFM pulling experiments of a-synuclein fibrils (see insert (i)). Pulling points (1–6) are indicated with red arrows in image (A) obtained before pulling. Image (B) was taken after pulling. Damaged sections of fibrils in image (B) are indicated with white arrows and numbered according to image (A). (C) Force curves for pulling the fibrils cross-linked to the APS-mica surface (trigger – 100pN, dwell time – 2s). The tip spring constant k=67.31 pN/nm. (D) Force curves for pulling of the non-covalently bound fibrils (trigger 500 pN, dwell time 2 s). The tip spring constant k=51.69 pN/nm. Pulling was done in PBS buffer.

Figure 8

AFM pulling experiments of a-synuclein fibrils (see insert (i)). Pulling points (1–6) are indicated with red arrows in image (A) obtained before pulling. Image (B) was taken after pulling. Damaged sections of fibrils in image (B) are indicated with white arrows and numbered according to image (A). (C) Force curves for pulling the fibrils cross-linked to the APS-mica surface (trigger – 100pN, dwell time – 2s). The tip spring constant k=67.31 pN/nm. (D) Force curves for pulling of the non-covalently bound fibrils (trigger 500 pN, dwell time 2 s). The tip spring constant k=51.69 pN/nm. Pulling was done in PBS buffer.

Figure 8

AFM pulling experiments of a-synuclein fibrils (see insert (i)). Pulling points (1–6) are indicated with red arrows in image (A) obtained before pulling. Image (B) was taken after pulling. Damaged sections of fibrils in image (B) are indicated with white arrows and numbered according to image (A). (C) Force curves for pulling the fibrils cross-linked to the APS-mica surface (trigger – 100pN, dwell time – 2s). The tip spring constant k=67.31 pN/nm. (D) Force curves for pulling of the non-covalently bound fibrils (trigger 500 pN, dwell time 2 s). The tip spring constant k=51.69 pN/nm. Pulling was done in PBS buffer.