Anthony L. Fink

Why are “natively unfolded” proteins unstructured under physiologic conditions?

“Natively unfolded” proteins occupy a unique niche within the protein kingdom in that they lack ordered structure under conditions of neutral pH in vitro. Analysis of amino acid sequences, based on the normalized net charge and mean hydrophobicity, has been applied to two sets of proteins: small globular folded proteins and “natively unfolded” ones. The results show that “natively unfolded” proteins are specifically localized within a unique region of charge‐hydrophobicity phase space and indicate that a combination of low overall hydrophobicity and large net charge represent a unique structural feature of “natively unfolded” proteins. Proteins 2000;41:415–427.

Protein aggregation: folding aggregates, inclusion bodies and amyloid

Aggregation results in the formation of inclusion bodies, amyloid fibrils and folding aggregates. Substantial data support the hypothesis that partially folded intermediates are key precursors to aggregates, that aggregation involves specific intermolecular interactions and that most aggregates involve beta sheets.

Disorder in the nuclear pore complex: The FG repeat regions of nucleoporins are natively unfolded

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of “natively unfolded” proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.

Pesticides directly accelerate the rate of α-synuclein fibril formation: a possible factor in Parkinson's disease

Parkinsons disease involves intracellular deposits of α‐synuclein in the form of Lewy bodies and Lewy neurites. The etiology of the disease is unknown, however, several epidemiological studies have implicated environmental factors, especially pesticides. Here we show that several pesticides, including rotenone, dieldrin and paraquat, induce a conformational change in α‐synuclein and significantly accelerate the rate of formation of α‐synuclein fibrils in vitro. We propose that the relatively hydrophobic pesticides preferentially bind to a partially folded intermediate conformation of α‐synuclein, accounting for the observed conformational changes, and leading to association and subsequent fibrillation. These observations suggest one possible underlying molecular basis for Parkinsons disease.

The association of alpha-synuclein with membranes affects bilayer structure, stability, and fibril formation.

The aggregation of α-synuclein is believed to be a critical factor in the etiology of Parkinsons disease. α-Synuclein is an abundant neuronal protein of unknown function, which is enriched in the presynaptic terminals of neurons. Although α-synuclein is found predominantly in the cytosolic fractions, membrane-bound α-synuclein has been suggested to play an important role in fibril formation. The effects of α-synuclein on lipid bilayers of different compositions were determined using fluorescent environment-specific probes located at various depths. α-Synuclein-membrane interactions were found to affect both protein and membrane properties. Our results indicate that in addition to electrostatic interactions, hydrophobic interactions are important in the association of the protein with the bilayer, and lead to disruption of the membrane. The latter was observed by atomic force microscopy and fluorescent dye leakage from vesicles. The kinetics of α-synuclein fibril formation were significantly affected by the protein association and subsequent membrane disruption, and reflected the conformation of α-synuclein. The ability of α-synuclein to disrupt membranes correlated with the binding affinity of α-synuclein for the particular membrane composition, and to the induced helical conformation of α-synuclein. Protofibrillar or fibrillar α-synuclein caused a much more rapid destruction of the membrane than soluble monomeric α-synuclein, indicating that protofibrils (oligomers) or fibrils are likely to be significantly neurotoxic.

A General Model for Amyloid Fibril Assembly Based on Morphological Studies Using Atomic Force Microscopy

Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.

Accelerated α-synuclein fibrillation in crowded milieu

Parkinsons disease is the second most common age‐related neurodegenerative disease, resulting from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of α‐synuclein has been implicated as a causative factor in the disease, and the process of fibril formation has been intensively studied in vitro with dilute protein solutions. However, the intracellular environment of proteins is crowded with other macromolecules, whose concentration can reach 400 g/l. To address this discrepancy, the effect of molecular crowding on α‐synuclein fibrillation has being studied. The addition of high concentrations of different polymers (proteins, polysaccharides and polyethylene glycols) dramatically accelerated α‐synuclein fibrillation in vitro. The magnitude of the accelerating effect depended on the nature of the polymer, its length and concentration. Our results suggest that the major factor responsible for the accelerated fibrillation under crowded conditions is the excluded volume.

Tau filaments from human brain and from in vitro assembly of recombinant protein show cross-β structure

Abnormal filaments consisting of hyperphosphorylated microtubule-associated protein tau form in the brains of patients with Alzheimers disease, Downs syndrome, and various dementing tauopathies. In Alzheimers disease and Downs syndrome, the filaments have two characteristic morphologies referred to as paired helical and straight filaments, whereas in tauopathies, there is a wider range of morphologies. There has been controversy in the literature concerning the internal molecular fine structure of these filaments, with arguments for and against the cross-β structure demonstrated in many other amyloid fibers. The difficulty is to produce from brain pure preparations of filaments for analysis. One approach to avoid the need for a pure preparation is to use selected area electron diffraction from small groups of filaments of defined morphology. Alternatively, it is possible to assemble filaments in vitro from expressed tau protein to produce a homogeneous specimen suitable for analysis by electron diffraction, x-ray diffraction, and Fourier transform infrared spectroscopy. Using both these approaches, we show here that native filaments from brain and filaments assembled in vitro from expressed tau protein have a clear cross-β structure.

Methionine oxidation inhibits fibrillation of human α-synuclein in vitro

We examined the effect of methionine oxidation of human recombinant α‐synuclein on its structural properties and propensity to fibrillate. Both oxidized and non‐oxidized α‐synucleins were natively unfolded under conditions of neutral pH, with the oxidized protein being slightly more disordered. Both proteins adopted identical partially folded conformations under conditions of acidic pH. The fibrillation of α‐synuclein at neutral pH was completely inhibited by methionine oxidation. This inhibitory effect was eliminated at low pH. The addition of oxidized α‐synuclein to the unoxidized form led to a substantial inhibition of α‐synuclein fibrillation.

Early Events in the Fibrillation of Monomeric Insulin

Insulin has a largely α-helical structure and exists as a mixture of hexameric, dimeric, and monomeric states in solution, depending on the conditions: the protein is monomeric in 20% acetic acid. Insulin forms amyloid-like fibrils under a variety of conditions, especially at low pH. In this study we investigated the fibrillation of monomeric human insulin by monitoring changes in CD, attenuated total reflectance-Fourier transform infrared spectroscopy, 8-anilinonaphthalenesulfonic acid fluorescence, thioflavin T fluorescence, dynamic light scattering, and H/D exchange during the initial stages of the fibrillation process to provide insight into early events involving the monomer. The results demonstrate the existence of structural changes occurring before the onset of fibril formation, which are detectable by multiple probes. The data indicate at least two major populations of oligomeric intermediates between the native monomer and fibrils. Both have significantly non-native conformations, and indicate that fibrillation occurs from a betarich structure significantly distinct from the native fold.