Temperature-Driven Stopped-Flow Experiments for Investigating the Initial Aggregation of the α-Synuclein Amyloid Protein, Focusing on Active and Inactive Phases.

The primary objective of this research is to further examine the events occurring during the active or burst phase by focusing on the aggregation of the Syn amyloid protein. Regarding this aspect, it was initially conducted rapid temperature variations using stopped-flow spectrometry and tyrosyl group fluorescence emission detection, within the initial 500 milliseconds in buffered Syn solutions at pH 7, exploring various temperature ranges to investigate protein aggregation. The results obtained were contrasted with results obtained for the N-acetyl-L-tyrosinamide (NAYA) parent compound in the same conditions.

Burst Phase Analysis of the Aggregation Prone α-synuclein Amyloid Protein.

While some studies inferred that valid information can be retrieved for the refolding of proteins and consequent identification of folding intermediates in the stopped-flow spectrometry collapse phase, other studies report that these burst phase folding intermediates can be questioned, implying a solvent-dependent modification of the still unfolded polypeptide chain. We therefore decided to investigate the burst phase occurring for the α-synuclein (Syn) amyloid protein by stopped-flow spectrometry. Solvent-dependent modification effects indeed occurred for the N-acetyl-L-tyrosinamide (NAYA) parent small compound and for the folded monomeric ubiquitin protein.

Initial Effect of Temperature Rise on α-Synuclein Aggregation - Entropic Forces Drive the Exposure of Protein Hydrophobic Groups Probed by Fluorescence Spectroscopy.

The aberrant formation of α-synuclein (Syn) aggregates, varying in size, structure and morphology, has been linked to the development of Parkinson's disease. In the early stages of Syn aggregation, large protein amyloid aggregates with sizes > 100 nm in hydrodynamic radius have been noticed. These low overall abundant large Syn aggregates are notoriously difficult to study by conventional biophysical methods.

Early α-synuclein aggregation is overall delayed and it can occur by a stepwise mechanism.

It is well-known that α-synuclein (Syn) protein aggregation is implicated in the pathogenesis of Parkinson's disease. There is an increased evidence that large protein aggregates populate very early the subsaturated solutions of several aggregate-prone proteins, including Syn. The role of these early large protein aggregates and the reaction processes that they involve remain elusive.

Identification of a biological excimer involving protein-protein interactions: A case study of the α-synuclein aggregation.

Excimer formation based on pyrene derivatives stacking has been used to probe conformational changes associated with a variety of protein interactions. Herein, in search for the nature of the protein interactions involved in amyloid proteins aggregation we studied the spectroscopic features of the N-acetyl-l-tyrosinamide (NAYA) parent compound and of a well-known aggregate amyloid protein, the α-synuclein (Syn). The aggregation of this amyloid disordered protein has been implicated in the development of Parkinson's disease, which is an increasingly prevalent and currently incurable neurodegenerative disorder.

Buffering capacity is determinant for restoring early α-synuclein aggregation.

For disordered proteins, including α-synuclein (Syn), the aggregation of which is implicated in Parkinson's disease, it is known that at mild acidic and at the pI solution conditions the use of either strong or weak electrolytes minimized Syn aggregation. The mechanism is driven by electrostatic forces but remains, however, poorly understood. To address this issue, we used two biological buffers as weak electrolytes, at a low concentration (10 mM) and monitored the aggregation of Syn solutions from pH 7 to pH 2, by means of light scattering techniques.

Evidence of the existence of micellar-like aggregates for α-synuclein.

We have been investigating the early stages of α-synuclein (Syn) aggregation, a small presynaptic protein implicated in Parkinson's disease. We previously reported that for pH jumps (1000 s) from pH 7 to pH 2 the variation of the Syn intrinsic fluorescence intensity did not change in the concentration range of ca. 10-50 μM (ref.

Interpretation of α-synuclein UV absorption spectra in the peptide bond and the aromatic regions.

The interpretation of the UV absorption spectra of proteins was a matter of intense debate in the second half of the last century. The study of the spectroscopic characteristics of peptide bonds in proteins was then of particular interest but the absorption of a large number of peptide bonds in a protein is a complex subject which gathers many contributions such as those from other amino acid residues that absorb as well and therefore unequivocal proofs remains a challenge. This probably becomes the reason for being an almost untouched subject of study in the last 40 years or so.

Earliest events in α-synuclein fibrillation probed with the fluorescence of intrinsic tyrosines.

The fluorescence of the four tyrosines of α-synuclein (Syn) was used for probing the earliest events preceding the fibrillation of Syn, during the onset of the so-called lag-time of fibrillation. Steady-state fluorescence experiments revealed an increase in the fluorescence intensity (FI) for Syn solutions at pH values 3 and 2, in comparison with pH7, and fluorescence decays indicated that the FI increase did not result from suppression of excited-state proton transfer from the tyrosines to aspartates and glutamates, exposure of tyrosines to more hydrophobic environments, or reduction of homo-energy transfer. Instead, the FI increase was due to changes in the population of the tyrosine rotamers at low pH values.

Mass spectrometric studies of the reaction of a blocked arginine with diketonic α-dicarbonyls.

The modification of arginine residues by diketonic α-dicarbonyls, in structural proteins and enzymes studies, is a process known for decades. The chemistry of these reaction processes is, however, not fully understood. Moreover, modification of arginine residues by reaction with α-dicarbonyls in glycation has also not been completely elucidated.

Reactions of aminoguanidine with α-dicarbonyl compounds studied by electrospray ionization mass spectrometry.

Aminoguanidine possesses extensive pharmacological properties. This drug is recognized as a powerful α-dicarbonyl scavenger. In order to better elucidate the reactivity of aminoguanidine with α-dicarbonyls, aminoguanidine was reacted with several aldehydic and diketonic α-dicarbonyls.

Heterogeneity and dynamics in the assembly of the heat shock protein 90 chaperone complexes.

The Hsp90 cycle depends on the coordinated activity of a range of cochaperones, including Hop, Hsp70 and peptidyl-prolyl isomerases such as FKBP52. Using mass spectrometry, we investigate the order of addition of these cochaperones and their effects on the stoichiometry and composition of the resulting Hsp90-containing complexes. Our results show that monomeric Hop binds specifically to the Hsp90 dimer whereas FKBP52 binds to both monomeric and dimeric forms of Hsp90.

Towards the control and inhibition of glycation-the role of the guanidine reaction center with aldehydic and diketonic dicarbonyls. A mass spectrometry study.

Glycation of proteins by glucose and formation of end-stage adducts (AGEs, advanced glycation end products) has been implicated in pathological mechanisms associated with diabetic complications, macrovascular disease, chronic and renal insufficiency, Alzheimer's disease, and aging. Of the carbonyl containing compounds involved in this process, alpha-dicarbonyls have particular importance, being established as direct intermediates in the formation of well-known AGEs. The guanidino group, present in arginine residues, suffers direct modifications by sugars and its derivatives, and is considered to be an important chemical basis, targeting the control and inhibition of glycation.

Non-enzymatic model glycation reactions--a comprehensive study of the reactivity of a modified arginine with aldehydic and diketonic dicarbonyl compounds by electrospray mass spectrometry.

Non-enzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes, and possibly aging and Alzheimer's disease. Among the amino residues in proteins arginine plays an important role, and its modification by sugar moieties generates the so-called advanced glycation end products (AGEs). Moreover, alpha-dicarbonyl compounds have been found as the main participants in those modifications.

Reactions of a modified lysine with aldehydic and diketonic dicarbonyl compounds: an electrospray mass spectrometry structure/activity study.

The phenomenon known as non-enzymatic glycation is described as the reaction of reducing sugars with basic amino groups of proteins and nucleic acids, as well as with simple amines, without enzyme mediation. Non-enzymatic model glycation reactions that make use of low-molecular-weight compounds make an important contribution in the elucidation of glicated processes in vitro and in vivo. Four alpha-dicarbonyl compounds, aldehydic (glyoxal, methylglyoxal and phenylglyoxal) and ketonic (diacetyl), were reacted with the modified amino acid N(alpha)-acetyl-L-lysine (AcLys) in an attempt to establish structure/activity relationships for the reactivity of alpha-dicarbonyls with the amine compound.