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Previous studies revealed that pesticides interact with α-synuclein and accelerate the rate of fibrillation. These results are consistent with the prevailing hypothesis that the direct interaction of α-synuclein with pesticides is one of many suspected factors leading to α-synuclein fibrillation and ultimately to Parkinson's disease. In this study, the biophysical properties and fibrillation kinetics of α-synuclein in the presence of rotenone were investigated and, more specifically, the effects of rotenone on the early-stage misfolded forms of α-synuclein were considered. The thioflavine T (ThT) fluorescence assay studies provide evidence that early-phase misfolded α-synuclein forms are affected by rotenone and that the fibrillation process is accelerated. Further characterization by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) shows that rotenone increases the amount of ordered secondary structure in this intrinsically disordered protein. Morphological characterization by transmission electron microscopy (TEM) and atomic force microscopy (AFM) provide visualization of the differences in the aggregated α-synuclein species developing during the early kinetics of the fibrillation process in the absence and presence of rotenone. We believe that these data provide useful information for a better understanding of the molecular basis of rotenone-induced misfolding and aggregation of α-synuclein.

Insulin has a largely alpha-helical structure and exists as amixture 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.

Synthetic amyloid beta-protein (A beta) is used widely to study fibril formation and the physiologic effects of low molecular weight and fibrillar forms of the peptide on cells in culture or in experimental animals. Not infrequently, conflicting results have arisen in these studies, in part due to variation in the starting conformation and assembly state of A beta. To avoid these problems, we sought a simple, reliable means of preparing A beta for experimental use. We found that solvation of synthetic peptide with sodium hydroxide (A beta x NaOH), followed by lyophilization, produced stocks with superior solubility and fibrillogenesis characteristics. Solubilization of the pretreated material with neutral buffers resulted in a pH transition from approximately 10.5 to neutral, avoiding the isoelectric point of A beta (pI approximately 5.5), at which A beta precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (A beta x TFA) or hydrochloric acid (A beta x HCl) salts of A beta, yields of "low molecular weight A beta" (monomers and/or dimers) were improved significantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that A beta x NaOH formed fibrils more readily than did the other A beta preparations and that these fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for the preparation of A beta for biophysical and physiologic studies.

A study examines the reactions taking place between several brain sphingolipids and [alpha]-synuclein, which is supposed to specifically bind to ganglioside GM1-containing small unilamellar vesicles (SUVs). The analysis reveals that the pertubration of GM1/raft association can lead to major changes in the [alpha]-synuclein that result in the pathogenesis of Parkinson's disease (PD), as it can be easily recruited to the caveolae and lipid fat regions in the membranes.

The presynaptic protein alpha-synuclein has four tyrosine and no tryptophan residues and hence a tyrosine is introduced to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. The results show the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.

The isolated B-chain peptide of bovine insulin is shown to form fibrils at both acidic or neutral pH. The effects of cross-seeding were examined to investigate the interaction between the two polypeptides. Results of the investigation demonstrate the significant 'species' barrier in amyloid fibril formation between fibrillation induced by different fibrils.

Abnormal filaments consisting of hyperphosphorylated microtubule-associated protein tau form in the brains of patients with Alzheimer's disease, Down's syndrome, and various dementing tauopathies. In Alzheimer's disease and Down's 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-[beta] 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-[beta] structure.

Research has been conducted on small acidic Ca (super)2+ -binding milk proteins, alpha-lactalbumin which form amyloid fibrils at low pH. Results indicate that fibril formation requires protein transformation into unfolded conformation.

Research has been conducted on Parkinson's disease causative factors, alpha-synuclein aggregation and fibrillation. The role of the glycosaminoglycans in the fibrillation of alpha-synuclein has been investigated and the results are presented.

Two point mutations, A53T and A30P, in the alpha-synuclein gene lead to self-association of the molecule as opposed to the wild-type. Data indicate that self-association in the mutants is due to an increased propensity for aggregation of the partially folded intermediates, which eventually leads to fibrillation of alpha-synuclein.

Research has been conducted on the insulin fibril formation. The study of the structural properties and kinetics of human insulin mutants' fibril formation to investigate the molecular basis of this formation is discussed.

Research shows that Caf1 and human interleukin-1beta are beta-structural proteins. The binding of Caf1 to interleukin-1 receptors of the mouse fibroblasts indicates adhesive properties of this protein, which may be involved in the bacteria gaining contact with the host.

Results demonstrate that the nucleation and insulin fibril growth are influenced by hydrophobic and electrostatic interactions. A model is presented describing the kinetics of the formation of critical nucleus leading to fibrils in response to experimental variables.

Research has been conducted on the light chain amyloids, a pathalogical condition in the kidneys. The properties of the recombinant amyloidogenic light chain variable domain have been investigated to determine whether aggregation can detect the partially folded intermediates.

Research has been conducted on the wild-type and a mutant staphylococcal nuclease. The salt-induced effects on the stability, structure and kinetics of staphylococcal nuclease attributed to the anion binding leading to the minimization of intramolecular electrostatic repulsion have been investigated.

Enzyme-induced strain-distortion in the ground-state ES complex in beta-lactamase catalysis has been shown by the use of Fourier transform infrared (FTIR) spectroscopy to exist. A major source of resistance to beta lactam antibiotics is beta-lactamases. Enzyme-facilitated hydrolysis caused by substrate distortion on binding to the enzyme is suggested by the very weak catalytic activity of the S70A beta-lactamase.

Research has been conducted on lysine 73. Mutation of lysine 73 to alanine in the beta-lactamase has been investigated.

Cytochrome c forms a dimer intermediate state during re-folding that has the properties of a molten globule. Imidazole was used to prevent misligated states from forming during re-folding.

Human prothymosin alpha (ProT alpha), an acidic nuclear protein, in its native unfolded state is discussed relative to its adoption of a partially folded collapsed conformation at acidic pH. Structural properties in the compact denatured state it assumes are different from those it has as a molten globule. The conformational changes may be associated with the protein activities.

Research was conducted to examine the conformational changes of an Hsp70 molecular chaperone induced by nucleotides, polypeptides, and N-ethylmaleimide (NEM). The structures of NEM-modified Ssa1p and Ssa1p were compared using a variety of biophysical methods. Results demonstrate that NEM alters the conformation of Ssa1p and disrupts, but does not eliminate, interdomain communication. Findings are consistent with a model wherein the lid is open in the presence of adenosine triphosphate but closed in the presence of adenosine diphosphate.

The equilibrium distribution of unfolded conformations of cytochrome c (Cty c) was studied using a small-angle x-ray scattering (SAXS) method. A cooperative transition is observed, with increasing denaturant and a similar Cm to that seen in circular dichroism. Scattering profiles showed that one state is native (N) and the other two states U(sub1) and U(sub2) are unfolded.

The photolysis reaction of CO-bound species was investigated via time resolved absorption and time-resolved circular dichroism (TRCD) methods to determine protein folding intermediates of cytochrome c. Results of TRCD studies revealed four intermediates with two, 50, 225 and 880 microsecond lifetimes. The findings further showed that folding after two microseconds may be controlled by litigation of His26/His33 using Fe(II).