Conformational distortion in a fibril-forming oligomer arrests alpha-Synuclein fibrillation and minimizes its toxic effects.

The fibrillation pathway of alpha-Synuclein, the causative protein of Parkinson's disease, encompasses transient, heterogeneous oligomeric forms whose structural understanding and link to toxicity are not yet understood. We report that the addition of the physiologically-available small molecule heme at a sub-stoichiometric ratio to either monomeric or aggregated α-Syn, targets a His50 residue critical for fibril-formation and stabilizes the structurally-heterogeneous populations of aggregates into a minimally-toxic oligomeric state. Cryo-EM 3D reconstruction revealed a 'mace'-shaped structure of this monodisperse population of oligomers, which is comparable to a solid-state NMR Greek key-like motif (where the core residues are arranged in parallel in-register sheets with a Greek key topology at the C terminus) that forms the fundamental unit/kernel of protofilaments. Further structural analyses suggest that heme binding induces a distortion in the Greek key-like architecture of the mace oligomers, which impairs their further appending into protofilaments and fibrils. Additionally, our study reports a novel mechanism of prevention as well as reclamation of amyloid fibril formation by blocking an inter-protofilament His50 residue using a small molecule.

Studies of cytochrome c-551 unfolding using fluorescence correlation spectroscopy and other biophysical techniques.

In this paper, we have studied the equilibrium unfolding transitions of cytochrome c from Pseudomonas aeruginosa (cytc551), a small bacterial protein. Similar to eukaryotic cytochrome c, cytc551 folds sequentially, although significant differences exist in the order of folding units (foldons). There are two regions of cytc551 (N-terminal helix with residue number 3 to 10 and the loop 2 region containing residues 34 to 45), in which no foldon unit could be assigned. In addition, the helix containing the Cys-X-X-Cys-His motif, adjacent to the N-terminal helix (residue number 3 to 10), shows unexplained ultra-fast collapse. To obtain further insights, we have studied cytc551 site-directed mutants using fluorescence correlation spectroscopy (FCS) and molecular dynamics simulation. We have found out that cytc551 unfolds through the formation of a fluorescently dark intermediate state and the amplitude of the dark component depends on the position of labeling. We have utilized this position dependence to propose a shape change model during the unfolding of cytc551. The present results show that the N-terminal helix remains in a collapsed position even in the completely unfolded state and this helix may act as a rigid support to guide the folding of its adjacent helix. This rigid support may be responsible for the ultra-fast collapse of the adjacent helix region, which occurs during the initial events of folding. The present results also show that the C-terminal end of loop 2 traverses a large distance during unfolding compared to the N-terminal end, which justifies the observed flexibility of the loop 2 region.

Molecular Crowding Affects the Conformational Fluctuations, Peroxidase Activity, and Folding Landscape of Yeast Cytochrome c.

To understand how a protein folds and behaves inside living cells, the effects of synthetic crowding media on protein folding, function, stability, and association have been studied in detail. Because the effect of excluded volume is more prominent in an extended state than in the native protein, a majority of these studies have been conducted in the unfolded state of different model proteins. Here, we have used fluorescence correlation spectroscopy (FCS) and other biophysical methods to investigate the effect of crowding agents Ficoll70 and Dextran70 on the nativelike state of cytochrome c from yeast. Yeast cytochrome c (y-cytc) contains a substantial expanded state in its native folded condition, which is present in equilibrium with a compact conformer in aqueous buffer. We have found that the crowding medium affects the native state equilibrium between compact and expanded states, shifting its population toward the compact conformer. As a result, the peroxidase activity of y-cytc decreases. Urea-induced protein stability measurements show that the compaction destabilizes the protein due to charge repulsions between similar charged clusters. Interestingly, the time constant of conformational fluctuations between the compact and expanded conformers has been found to increase in the crowded milieu, suggesting a crucial role of the solution microviscosity.

Subtle Change in the Charge Distribution of Surface Residues May Affect the Secondary Functions of Cytochrome c.

Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c.

Conversion of amyloid fibrils of cytochrome c to mature nanorods through a honeycomb morphology.

Amyloid species with various morphologies have been found for different proteins and disease systems. In this article, we aim to ask if these morphologies are unique to a particular protein or if they convert from one to another. Using a heme protein containing iron as the transition-metal activator of aggregation and a negatively charged surfactant, partial unfolding of the protein and its aggregation have been induced. In the pathway of aggregation, we have observed the formation of several morphological structures of a single protein, which were visualized directly using atomic force microscopy (AFM). These structures have been found to appear and disappear with time, and their formation could be monitored under normal buffer conditions and at room temperature without requiring any sophisticated chemical or biological methodologies. In addition, we have observed the formation of honeycomb-shaped morphology, which may serve as an intermediate. These amyloid-based nanostructures may have the potential to be explored in therapeutics delivery and other biomedical applications.

Micellization of cetyltrimethylammonium bromide: effect of small chain Bola electrolytes.

Sodium dicarboxylates (or Bola salts) with methylene spacers 0, 2, 4, 6, 8, and 10 were studied in aqueous solution to investigate their influence on the micellization of cetyltrimethylammonium bromide (CTAB). Since bolas with spacer length ≤12 are known not to micellize in general, the herein used sodium dicarboxylates were treated as 2:1 amphiphilic electrolytes which reduced surface tension of water (except sodium oxalate with zero spacer) without self-association. Their concentration dependent conductance was also linear without breaks. The bolas affected the micellization of CTAB but acted like salts to decrease its CMC. Their combinations did not form bilayer aggregates as found in vesicles. Nevertheless, they synergistically interacted with CTAB at the air/water interface as revealed from Rosen's thermodynamic model. Hydrodynamic radius (Rh), Zeta-potential (ζ), and electrical double layer behavior of bola interacted CTAB micelles were assessed. From SANS measurements, micelle shape, shape parameters, aggregation number (Nagg), surface charge of the bola influenced CTAB micelles were also determined. NMR study as well supported the non-mixing of bolas with the CTAB micelles. They interacted in solution like "amphiphilic electrolytes" to influence the surface and micelle forming properties of CTAB.