Search

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.

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.

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.

It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in a monomeric, partially folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations. In addition, more globularity, as determined from Kratky plots of small-angle x-ray scattering data, was also noted in the associated states.

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).

Three slow kinetic phases describe the low-temperature fluorescence-detected refolding of staphylococcal nuclease (SNase). Peptidyl prolyl cis-trans isomerase, which is proven to catalyze the slow folding reactions of some proteins, was used in determining which of the refolding reactions of SNase and P117G SNase involve proline isomerization. Results support the hypothesis that the slow folding steps involve isomerization of non-native cis proline peptide bonds are are tightly coupled to denaturant-sensitive structural changes.

[Beta]-Lactamases are the primary cause of [Beta]-lactam antibiotic resistance in many pathogenic organisms. The [Beta]-lactamase catalytic mechanism has been shown to involve a covalent acyl-enzyme. Examination of the structure of the class A [Beta]-lactamase from Bacillus licheniformis suggested that replacement of Asn-170 by leucine would disrupt the deacylation reaction by displacing the hydrolytic water molecule. When N170L [Beta]-lactamase was reacted with penicillins, a novel product was formed. We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine-oxazolinone as the initial product. The oxazolinone subsequently undergoes rapid breakdown leading to the formation of N-phenylacetylglycine and N-formylpenicillamine. This appears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered product, effectively changing the product specificity of [Beta]-lactamase into that of D-Ala-D-Ala-carboxypeptidase interacting with benzylpenicillin.

The conformational and structural characterizations of the heat shock protein DnaK were determined by small-angle X-ray scattering (SAXS) and size-exclusion chromatography (SEC). DnaK has a dumbbell-shaped structure as evidenced by its bimodal P(r) distribution function, biphasic Guinier and Kratky plots. The association state of the protein is affected by the buffer concentration. DnaK dimerizes at high buffer and protein concentrations resulting to an increase in its measured radius of gyration and maximum dimension.

Mutagenesis of Glutamate-166 in Bacillus licheniformis to aspartate and cysteine results in a branched-path mechanism that involves a shift in the kinetic mechanism from the linear, acyl-enzyme pathway to an inactive form. The shift results from a hydrolysis rate reduction of the normal acyl-enzyme. The branched pathway is specific to beta-lactamase catalysis during low deacylation. Glu-166 acts as a base catalyst during generation and hydrolysis of the acyl-enzyme intermediate.

1, 1'-Bis(4-anilino-5-naphthalenesulfonic acid) (Bis-ANS), a hydrophobic fluorescent molecular probe that binds to protein intermediate states and to nucleotide binding sites, binds selectively to the partially folded intermediate state (I) and consequently induces an equilibrium shift from the native state to the I state. Bis-ANS binds to the native state of Dna K under conditions such as low dye concentration, nucleotide presence and a temperature less than the free energy temperature of the protein.

Attenuated total reflectance facilitated the study of the secondary structure of the human interleukin-1 beta that occurred in aggregated forms such as inclusion bodies. Beta-sheets constituted 52% of the total structure while turns occupied 23%. The remaining 25% consisted of either a loop or an irregular structure.

Fourier transform-infrared spectroscopy analysis of thermally denatured RNAse A reveals unstable quantities of secondary structures of a compact nature. Thermal denaturation greatly reduces the amount of beta-sheet and alpha-helix while increasing turn and unordered structure amounts. The results are not in agreement with other studies indicating the absence of stable hydrogen-bonded structures under similar conditioning.

Differing conditions of pH and salts are used to calculate the structure of apocytochrome c and a fluorescence-labeled product. An intermediate position, called the A position, is adopted by the apocytochrome c as hypothesized, when anions occur at acidic pH levels. The A position is similar to the molten globule of the holoprotein. A rise in the pH revealed a reduction in the constancy of the A position. A concise denatured occurrence is preferred by a fall in the total charge, although the constancy of the molten globule state cannot be obtained by this method. The surroundings influence the ability of apocytochrome c to take on unique concise denatured structures.

Bacillus licheniformis is similar to Bacillus cereus at low pH despite differing isoelectric points. The replacement of Glu-166 by Cys has no effect on the conformational properties of beta lactamase. The spin label mobility in the A state is similar to that in the native state, though in the unfolded state it is less mobile. At low pH with consistent molten globule properties, beta lactamase from bacillus licheniformis forms a moderate salt concentration (A state) and a stable compact intermediate state of cytochrome. The intermediate state is considered to be a molten globule, a compact state containing great amounts of secondary structure. The mobility of the spin level decreases with aggregation involving protein and protein interactions.

The guanidine hydrochloride induced unfolding of the heat shock protein DnaK was studied using flourescence, size-exclusion HPLC, circular dichroism and dynamic light scattering. The denaturation process was noted to occur in two stages with one expandable intermediate state in between. The first stage was influenced by the presence of Mg/ADP or Mg/ATP although the next transition was unaffected by nucleotides. Thus, a hierarchical system of unfolding was suggested to characterize the denaturation of DnaK.

The effects of urea on apomyoglobin solubility were investigated. It was found that apomyoglobin precipitation is a thermodynamically reversible process independent of the pathway of aggregation. At low urea concentrations, the solubility of apomyoglobin decreases with an increase in urea concentration. The results show that the primary aggregating species are denatured protein molecules, rather than folding intermediates.

A study was conducted on the effect of proline on folding stability and kinetics in wild-type staphylococcal nuclease and its mutants. Slight increases in stability were observed when Gly or Thr were substituted for Pro-117, while slight decreases were obtained when Pro-31 was replaced by Ala. Trytophan fluorescence studies revealed that for P31A and the wild-type protein, refolding kinetic traces were triphasic, while they were biphasic for the P117G and P117T mutants. Mutations had no effect on the rates of the wild type's fastest and medium phases. It was concluded that Pro-117 isomerization causes the guanidine-independent slow-folding kinetic phase in the wild type.

Hsp60 and Hsp70, the molecular chaperone proteins, bind themselves to short peptides and unfolded proteins when twisted in the cell functions under stress induced and normal conditions. The disintegration of the substrate polypeptides from the chaperone is caused by the addition of Mg-ATP, and occurs since the ATP analogue is not capable of replacing ATP. The disintegration Hsp 70-substrate proteins require the hydrolysis of the Mg-ATP in addition to the earlier mentioned reactions. Mg-ATP binding is more essential than the Mg-ATP hydrolysis to the disintegration of the molecular chaperone proteins.

The article studies the molecular mechanism of the flavonoid-induced inhibition of [alpha]-synuclein fibrillatio using various biochemical and biophysical approaches. Findings reveal that flavonoids inhibiting [alpha]-synuclein fibrillation could serve as a model for the development of therapeutic drugs in combating Parkinson's disease.

The effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-[alpha]-lactalbumin, human insulin, bovine core histones and human [alpha]-synuclein, is analyzed. The effect of crowding agents on the aggregation patterns under a variety of conditions favoring different aggregated end products in diluted solutions is analyzed in order to study how macromolecular crowding has guided protein between different aggregation pathways.

A study was conducted to determine the changes induced by methionine oxidation in the structural properties, conformational stability, and aggregation behavior of immunoglobulin light chain domain LEN. The results revealed that the LEN oxidation might play an important role in amorphous deposition of the protein but not in its fibrillation.

The structural impact of three different parkinsonism-associated oxidation stress response protein (DJ-1) missense mutations (A104T, E163K, and M26I) is described. The missense mutations are located near the junction between secondary structural elements in DJ-1 and are implicated in reducing the thermal stability of DJ-1 in solution.

Studies are conducted with alpha-synuclein and three mutants to determine the roles of the different regions of alpha-synuclein in the assembly of fibrils. Results indicate that the terminal regions of alpha-synuclein do not participate in the assembly of the protofilament core, but are crucial for the interactions between protofilaments, necessary for fibril maturation.

The aggregation of the small, ample, essentially disordered presynaptic protein [alpha]-synuclein is supposed to be a significant step in Parkinson's disease. The detailed examination into the molecular basis for [alpha]-synuclein aggregation/fibrillation along with the factors that increase or hamper fibrillation, effects of molecular crowding, oxidation, point mutations, and lipid membranes, and different conformational and oligomeric states adopted by [alpha]-synuclein is presented.

An examination on whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH is conducted. Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions.

The effect of [alpha]-synuclein on lipid oxidation in membranes containing phospholipids with unsaturated fatty acids is investigated. The findings suggest that the inhibition of lipid oxidation by [alpha]-synuclein is a physiological function of the protein.

A study was conducted to analyze the structural and fibrillation properties of a series of alpha-synuclein mutants. The result shows that the degree of inhibition of fibrillation by MetO alpha-synuclein is proportional to the number of oxidized methionines.

The study shows that anions induce partial folding of alpha-synuclein at neutral pH, forming a critical amyloidogenic intermediate, which leads to significant acceleration of the rate of fibrillation. It concluded that anions promote fibrillation of alpha-synuclein through a combination of electrostatic and solvent effects.

Results show that dissociation and transformation of insulin hexamers into dimers and partially unfolded dimers and monomers occurs with varying concentrations of guanidine hydrochloride, with high concentrations yielding mostly unfolded monomers with some residual structure. Further, low concentration of guanidine hydrochloride significantly accelerates fibrillation conforming to the partially folded state of insulin monomers.

The authors have investigated alpha-synuclein aggregation. They demonstrate that alpha-synuclein forms a histone complex, and that the presence of histones increases alpha-synuclein's fibrillation rate.

Results demonstrate that fibrils are formed from the oligomers of conformationally less stable LEN immunoglobulin either directly or by dissociation. Data show structural rearrangements with resultant increase in disordered secondary structure, solvent accessibility, and a decrease in intrinsic stability of the soluble oligomer intermediates.

The effect of membrane environments on alpha-synuclein is investigated by analyzing structural and fibrillation characteristics of the protein in mixtures of water and fluorinated alcohols. Results reveal that all solvents induce folding of alpha-synuclein, eventually leading to fibrillation. High concentrations of organic solvents inhibit protein fibrillation. These conformational states mimicking phospholipid vesicles explain membrane induced fibrillation of alpha-synuclein.

Aggregation of Ig light chains to form amyloid fibrils is a characteristic feature of light-chain amyloidosis, a light-chain deposition disease. A recombinant variable domain of the light chain SMA was used to form amyloid fibrils in vitro. Fibril formation was monitored by atomic force microscopy imaging. Single filaments 2.4 nm in diameter were predominant at early times; protofibrils 4.0 nm in diameter were predominant at intermediate times; type I and type II fibrils 8.0 nm and 6.0 nm in diameter, respectively, were predominant at the endpoints. The increase in number of fibrils correlated with increased binding of the fluorescent dye thioflavin T. The fibrils and protofibrils showed a braided structure, suggesting that their formation involves the winding of protofibrils and filaments, respectively. These observations support a model in which two filaments combine to form a protofibril, two protofibrils intertwine to form a type I fibril, and three filaments form a type II fibril.

I. INTRODUCTION The basic paradigm of molecular chaperones is that they recognize AND selectively bind nonnative, but not native, proteins to form relatively stable complexes (48). In most cases, the [...]