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 Nα-acetyl-L-tyrosinamide (NAYA) parent small compound and for the folded monomeric ubiquitin protein. More complex was the burst phase analysis of the disordered Syn amyloid protein. While this amyloid protein was determined to be aggregated at pH 7 and pH 2, in particular, this protein at pH 3 appears to be in a monomeric state in the burst phase analysis performed. In addition, the protein at pH 3 appears to suffer a hydrophobic collapse with the formation of a possible folded intermediate. This folded intermediate seems to result from a fast contraction of the disordered amyloid polypeptide chain, which is proceeded by an expansion of the protein, due to the occurrence of solvent-dependent modification effects in a milliseconds time scale of the burst phase. Generally, it can be argued that both literature criteria of solvent-dependent modifications of the disordered Syn amyloid protein and of the formation of its possible folded intermediate are very likely to occur in the burst phase.

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. Due to the growing importance of studying the early stages of Syn aggregation, we developed a strategy to achieve this purpose, which is the study of the initial effect of the Syn protein aqueous solutions temperature rise. Therefore, the increase of the Syn aqueous solutions entropy by the initial effect of the temperature rise led to the exposure of the protein hydrophobic tyrosyl groups by not interfering with this amyloid protein aggregation. As an attempt to interpret the degree of the referred protein tyrosyl groups exposure, the classic rotameric conformations of the Nα-acetyl-L-tyrosinamide (NAYA) parent compound were used. For both NAYA and Syn, it was determined that the classic rotameric conformations involving the tyrosyl groups indeed accounted for their exposure under steady-state conditions of fluorescence, for lowest molecular species concentrations investigated at least. In this situation, Syn aggregation was observed. For the higher NAYA and Syn concentrations studied, the referred classic rotameric conformation were insufficient in such referred steady-state conditions and, for Syn, in particular, fluorescence anisotropy measurements revealed that less protein aggregation occurs along with its delay. Overall, the developed strategy by focusing on the initial effect of the temperature rise of Syn aqueous solutions in lower concentrations is suitable for informing us about the degree of this protein aggregation in solution.

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. Amyloid protein's fluorophores (aromatic residues) can retrieve information regarding the amyloid protein's aggregation, by monitoring their fluorescence intensity. By excitation of Syn tyrosine residues in a low ionic strength medium (0.01 M tris-HCl) and collecting the time resolved fluorescence (stopped-flow analysis) it was possible to discriminate a time window of the first ca. 2 s, corresponding to the prevalent dissociation of early large Syn aggregates formed. Lowering even further the media ionic strength, such as Syn in water and Syn in solution containing 1,4-dioxane (pH ≈ 6.5), the above referred time window of the first ca. 2 s was abolished. It should be expected that Syn aggregation mainly occurred. In fact, Syn aggregation is initially delayed by the addition of a structure-induced agent (1,4-dioxane) in a stepwise mechanism. This study retrieves that very early the large Syn aggregates formed are unstructured and, in low ionic strength media (>0.01 M), they restructure in the dissociation process and intertwined the occurrence of its aggregation. In lower ionic strength media (<0.01 M), the large Syn aggregates dissociation is abolished and its aggregation is initially delayed, conferring to these protein aggregates restructuring in a stepwise mechanism.

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. Aspects related to the early stages of the condensation of arginine with one dicarbonyl molecule, leading to the formation of dihydroxyimidazolidines and their dehydrated forms, in particular, are here approached in more detail. Taking into consideration the usually rapid kinetics involved in the formation of the early reaction product species, we decided to use fast, sensitive and selective analytical techniques, such as electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS(n)) to monitor the reactions of a blocked arginine (acetyl-arginine) with several selected diketonic α-dicarbonyls, to identify and characterize the mentioned transient species and to probe the reaction mechanism involved. Compounds grouped into two different classes according to their structural similarity were identified, namely acetyl-dihydroxyimidazolidines and acetyl-bis(dihydroxyimidazolidines), together with their dehydrated species. The former compounds are known to exist in solution. The reactivity of acetyl-bis(dihydroxyimidazolidines) seems to be different from that of acetyl-dihydroxyimidazolidines. To note that dehydration appears to be reinforced in acetyl-bis(dihydroxyimidazolidines) chemistry with respect to acetyl-dihydroxyimidazolidine chemistry, while both structurally related compounds involve mostly dihemiaminals reactivity. Two different ion structures are proposed for single dehydrated acetyl-bis(dihydroxyimidazolidines), concerning the two more symmetrical and two more asymmetrical dicarbonyls reacted. In acetyl-bis(dihydroxyimidazolidines) formation, we concluded that the importance of single dehydration relies on the rapid minimization of sterics and energetics of the reaction moieties formed. These reactions occur also in a selective way, regarding the two compound structures proposed for single dehydrated acetyl-bis(dihydroxyimidazolidines). Further considerations are also established for the formation of single dehydrated acetyl-bis(dihydroxyimidazolidines). An explanation for the reversible nature of the reaction of arginine with diketonic dicarbonyls is also provided. This study reinforces the potential of the fast, sensitive and selective electrospray ionization mass spectrometry techniques for the investigation of transient species and their mechanistics, that might otherwise not be feasible by means of the most commonly used spectroscopic techniques.

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. By preforming Hsp90 complexes with either Hop, followed by addition of FKBP52, or with FKBP52 and subsequent addition of Hop, we monitor the formation of a predominant asymmetric ternary complex containing both cochaperones. This asymmetric complex is subsequently able to interact with the chaperone Hsp70 to form quaternary complexes containing all four proteins. Monitoring the population of these complexes during their formation and at equilibrium allows us to model the complex formation and to extract 14 different K(D) values. This simultaneous calculation of the K(D)s from a complex system with the same method, from eight deferent datasets under the same buffer conditions delivers a self-consistent set of values. In this case, the K(D) values afford insights into the assembly of ten Hsp90-containing complexes and provide a rationale for the cellular heterogeneity and prevalence of intermediates in the Hsp90 chaperone cycle.

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. Electrospray ionization mass spectrometry is a suitable technique to study chemical and biochemical processes, and was selected for the purpose. In aminoguanidine reactions, triazines were detected and, other compounds that have never been reported before were identified. Triazine precursor forms were detected, namely tetrahydrotriazines and singly dehydrated tetrahydrotriazines. Moreover, species with bicyclic ring structures, and dehydrated forms, were also identified in aminoguanidine reactions. These species appear to result from tetrahydrotriazines and triazines reactions with one dicarbonyl molecule. Experiments revealed that these bicyclic species, in particular the ones resulting from triazines reactivity, could exist in solution, since they were both identified in the reactions of aminoguanidine and of a selected triazine with the dicarbonyls studied. The results obtained, regarding aminoguanidine/triazines reactivities, appear to support the capability of triazines to condensate and form polycyclic ring structures, and also to support literature mechanistic data for dihydroimidazotriazines formation via dihydroxyimidazolidine-triazines. The data obtained in this study may prove to be valuable to complement solution information, concerning the reactivity of amines with α-dicarbonyls, in particular.

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 Nα-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. Also, Syn aggregation has been widely investigated but, information concerning the conformational alterations in the diverse protein aggregated species at the molecular level, is still scarce. Three different molecular configurations of the NAYA parent compound were at least found to exist in its solutions containing 1,4-dioxane. Two of these NAYA molecular configurations were found to produce a more efficient excimer fluorescence. For Syn solutions containing 1,4-dioxane, one molecular configuration involving the intermolecular interaction between the protein tyrosyl group and the protein peptide bond was found to exhibit excimer fluorescence. This study is the first one reporting the formation of a biological excimer exhibiting fluorescence. Although very weak, this can be used as a signature of protein-protein interactions and, ultimately, enabling to access the complex interactions network existing in the amyloid aggregated species.

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. When the citrate buffer was used, in which there is buffering capacity in the pH range studied, the maximum of Syn aggregation was very close to the isoelectric point (pI = 4.7). When using tris-HCl, in which there is almost no buffering capacity in the pH range studied, it was for the first time observed a slow transition of the pI (of ca. 1 h) from 4.7 to 4-3, for a 33.5 µM protein concentration, as an example. We also observed in the protein solutions (in tris-HCl) the very early formation of large Syn aggregates. When there is buffering capacity, such as pH 7, these early large Syn aggregates dissociate, followed by association/aggregation. When there is no buffering capacity, such as pH 3, the referred early large Syn aggregates only dissociate. Overall, early large Syn aggregates dissociation can cause entropy in the protein solutions and Syn aggregation is only restored by the altered electrostatic forces due to the existing buffering capacity. Finally, by using an innovative strategy based in the ANS dye fluorescence intensity variation, we determined of the occurrence of the liquid-liquid phase separation process at pH 7 Syn solutions.

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. 16). Additionally, I reported dynamic light scattering (DLS) experiments revealing the formation of early large Syn aggregates (ref. 7). These reported results mean that some molecular entity is being early formed. Herein, it was decided to investigate in detail these early Syn aggregates by using light scattering. By DLS analysis, these aggregates exhibited a hydrodynamic diameter of ca. 420 nm along with a high scattering intensity, characteristic of micellar-like aggregates formation. The critical micelle concentration (CMC) at which the Syn micellar-like aggregates are formed was ca. 10 µM. DLS analysis has also revealed that the micellar-like aggregates for Syn evolved, for protein concentrations >100 µM, to the formation of smaller aggregates (hydrodynamic diameter of ca. 165 nm), possibly Syn oligomers. The Syn micellar-like aggregates formed at pH 7 solutions seem to be active species and to have a role in this protein aggregation mechanism.

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. In this report the spectroscopic characteristics of the amyloid disordered protein α-synuclein (Syn) were studied in detail, concerning the UV absorption spectra in the peptide bond (200-230 nm) and the aromatic regions. Several protein concentrations, several solution pH and the first 300 min of the aggregation reaction were here investigated. In what the peptide bond region of Syn is concerned, UV difference spectra for a 33.5 µM protein solution concentration, in particular, revealed that at Syn solutions pH 7, 3 and 2 the counter-ion concentration increases in that order, as expected, accounting for the decrease of the peptide bond absorbance. Also, for this protein solution concentration, quantitative information can be obtained from peptide bond absorption and counter-ion concentration interplay in what the progression of the Syn aggregation reaction is concerned. This situation represents a label-free analysis of Syn aggregation in the lag-phase, in particular. Concerning the aromatic region of Syn, the UV absorption spectra revealed a perturbation at ca. 290-310 nm which is not related with light scattering effects in the UV absorption spectra but is related with the formation of mostly intermolecular hydrogen-bonded complexes between Syn tyrosyl groups and aspartic and glutamic acids residues. Interestingly, is the possible enrollment of these intermolecular complexes in the stabilization of this highly dynamic disordered protein in solution.

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. Four alpha-dicarbonyl compounds, aldehydic and ketonic, were reacted with the modified amino acid N(alpha)-acetyl-L-arginine (AcArg), in an attempt to establish structure/activity relationships for the reactivity of alpha-dicarbonyls with the amine compound. Electrospray ionization mass spectrometry (ESI-MS), combined with tandem mass spectrometry (MS/MS), was used to identify and characterize reagents, intermediates and reaction products. The fragmentation patterns of precursor ions showed similarities in all reaction systems studied, in which fragmentation of the amino acid residue prevails, especially for the dehydrated and/or multiple dehydrated precursor ions. For the non-hydrated ion species, fragmentation of the arginyl guanidino group was mainly observed. Specific information regarding the nature of the ions formed, in which the dicarbonyl electrophile character played an important role, was obtained. As an example, singly and doubly hydrated acetyl-argpyrimidine ions were detected for the methylglyoxal reaction only. For symmetrical dicarbonyls, glyoxal and diacetyl, the importance of steric contributions with respect to the energetic ones is discussed. Furthermore, the dehydrated acetyl-tetrahydropyrimidine ions for methylglyoxal and phenylglyoxal reactions revealed fragment ion compositions including the protonated molecules of acetyl-argpyrimidine, -hydroimidazolone and -5-methylimidazolone. An explanation for the acetyl-argpyrimidine formation from the acetyl-hydroimidazolone formation reaction is proposed. Aspects such as the amount of acetyl-hydroimidazolone formed, the response of the hydration equilibria of the dicarbonyl forms to the new unhydrated dicarbonyls introduced by the reversal of the acetyl-hydroimidazolone formation reaction and the stability of the dicarbonyl intermediate involved in the acetyl-argpyrimidine formation are proposed, as being responsible to control the formation of acetyl-argpyrimidine.

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. Seven dicarbonyl compounds, aldehydic and diketonic, were reacted with guanidine, in an attempt to establish structure/activity relationships. Electrospray mass spectrometry, together with tandem mass spectrometry, was used to identify and characterize the reaction products. The reactivity of guanidine was found to vary with the dicarbonyls used. For glyoxal, a high amount of dihydroxyimidazolidine was formed, whereas for methylglyoxal, dihydroxyimidazolidine was slowly converted into hydroimidazolone. Interestingly, aqueous guanidine was found to prevent argpyrimidine formation. The formation of several amine-dicarbonyl moieties was observed for the larger alkyl-diketonic dicarbonyls reaction systems, in particular. Molecular structures, bearing a polar chain, of an imidazole ring, and a nonpolar one, of alkyl groups, located at both sides of the imidazole rings, were attributed to these moieties. Gas-phase experiments suggested that the larger alkyl groups have a preference for being located at one of the sides of the imidazole rings. Moreover, the referred amine-dicarbonyl moieties are formed via (dihydroxyimidazolidine - 2H2O) moieties. The latter (dihydroxyimidazolidine - 2H2O) moieties are formed in high amounts in the larger alkyl-diketonic dicarbonyl reactions. Since these moieties react with dicarbonyl molecules, and react even faster with already modified amine functions, we can foresee that these species may be useful for controlling and inhibiting glycation of larger biomolecules, such as proteins.

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. Electrospray ionization mass spectrometry (ESI-MS) combined with tandem mass spectrometry (MS/MS) and collision-induced dissociation (CID) was used to identify and characterize reagents, intermediates and reaction products. The formation of dicarbonyl-derived lysine dimers was observed exclusively. Especially, attention is drawn to alkyl- (asymmetrical dicarbonyl systems) and carboxyl- (glyoxal system) substituted imidazolium ions, at ring position 2. The main differences observed in the reactions studied were related to the reactivity with the diimine intermediate. This intermediate can react either with a non-hydrated dicarbonyl molecule at the aldehydic carbonyl, or with a mono-hydrated one at the ketonic carbonyl, particularly for asymmetrical dicarbonyls. For 2-carboxyl-substituted imidazolium ion (glyoxal reaction), besides the usual keto-enol rearrangement from the diol group, an alternative reaction pathway (proton abstraction) appears to contribute also for the imidazolium ring-closure process. Moreover, the formation of imidazolium ring structures can depend on several factors, namely, the presence (or absence) of electron donor substituents at the formed diol, the degree of stability of the new electrophile generated and/or the equilibrium concentration of the non- and mono-hydrated dicarbonyl forms in solution, the last being particularly important for asymmetrical dicarbonyls. The results reported reveal the complexity of reactivity as well as the diversity of imidazolium molecular structures.

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. Stopped-flow experiments (pH-jumps) showed that the FI enhancement involves two processes: a fast (sub-7 ms) intramolecular (concentration-independent) process, which we assign to the protein collapse at low pH, and a slower intermolecular (concentration-dependent) process of protein dimerization/oligomerization, starting at 4-10s after acidification. To the best of our knowledge, this is the first work on the experimental detection of these earliest processes in the fibrillation of Syn.