Marco António Gomes Saraiva
@tecnico.ulisboa.pt
RESEARCH INTERESTS
Biophysics
Scopus Publications
Scopus Publications
Marco A. Saraiva and M. Conceição Oliveira
Elsevier BV
Marco A. Saraiva
SAGE Publications
Currently, there is increasing interest in identifying the mechanistic characteristics of the α-synuclein amyloid protein aggregation during its early stages. The initiation of amyloid protein incubation was investigated by applying the concepts of hydrophobic hydration in the early-formed protein aggregates and the light transport in the protein samples by using near-infrared light. These are unexplored concepts in amyloid protein aggregation research. Early-formed protein aggregates develop solvent-exposed hydrophobic residue segments, and intramolecular and intermolecular interactions can be identified by hydrophobic hydration, while consecutive intramolecular interactions can cancel this effect. In the light transport within protein samples, at low protein concentrations, the early-formed protein aggregates achieve stability, whereas at higher concentrations, such as those found in neuronal synapses (∼50 µM), the early-formed aggregates continue to develop.
Marco A. Saraiva
Springer Science and Business Media LLC
AbstractThe 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 Nα-acetyl-L-tyrosinamide (NAYA) parent compound in the same conditions. The utilization of the NAYA compound is suitable as it mimics the peptide bonds in proteins and contains a tyrosyl group resembling the four tyrosyl groups found in the Syn protein structure (the protein has no tryptophan residues). Furthermore, the NAYA compound adopts an intramolecularly hydrogen-bonded structure even in an aqueous solution, similar to the interactions seen in the hydrophilic face of β-sheets. Additionally, the Syn protein system can exhibit the presence of β-sheets as a result of the existence of very low abundant Syn amyloid precursor forms or nuclei during the initial stages of the protein aggregation. Thus, a relationship is present between the molecular processes in the NAYA and Syn protein systems, making the NAYA’s application crucial in this research. Moreover, to aid in understanding the results, it was also compared the events during the quiescent or inactive phase (30–500 milliseconds) with those in the burst phase (up to 10 milliseconds) using stopped-flow spectrometry conditions. Steady-state measurements were beneficial in comprehending the occurrences in both the quiescent and burst phases examined. Although protein aggregation and disaggregation were observed during the quiescent phase, determining these processes in the burst phase was more challenging. In the latter case, the aggregation of the Syn protein is actually initiated by the interaction of the intrinsically disordered Syn monomers. In the quiescent phase, first-order rate constants were measured and analysis showed that Syn protein aggregation and disaggregation occur simultaneously. At lower temperatures, early protein disaggregation outweighs protein aggregation whereas at higher temperatures protein disaggregation and aggregation are rather similar. It is also need to highlight that the burst phase, while distinct from the quiescent phase, can be considered as a possible structural phase for obtaining details about the aggregation of this specific disordered protein in solution on a very short timescale.
Marco A. Saraiva
Springer Science and Business Media LLC
Abstract Currently, there is an increased interest in identifying the characteristics of amyloid aggregates in the initial stages of amyloid formation. The aggregation mechanism of the α-synuclein (Syn) amyloid protein, which has been extensively studied, is still not fully understood. I show that with conventional dynamic light scattering (DLS) technique, the measurements of the dimensions of Syn amyloid precursor forms can be done early in the protein incubation. Additionally, the early aggregation of the Syn protein was initially studied by analyzing autocorrelation functions from fit distributions up to 104 µs in the initial DLS measurements, specifically within the first 21 min. Investigation was conducted on the variation in the pH of the Syn solution throughout time. Based on DLS data, large Syn aggregated species formed from the monomer protein species. Afterward, I generated the autocorrelation functions based on the original DLS data, extending the fit distributions up to 105 µs and noticed the existence of elongated Syn amyloid precursor forms in the protein solutions. Because the length of the elongated Syn amyloid precursor forms closely matches the wavelength of the incident light, the combination of translational diffusion Dt and rotational diffusion Dr in the decay rates enabled the measurement of their geometric dimensions through DLS. The improved precision of the fitted distributions I offered resulted in a new interpretation for the Syn protein aggregation in the initial stages. Overall, the methodology used in this study could be an effective strategy for examining how Syn amyloid precursor forms develop over time.
Marco A. Saraiva and M. Helena Florêncio
Springer Science and Business Media LLC
AbstractWhile 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.
Marco A. Saraiva and M. Helena Florêncio
Springer Science and Business Media LLC
AbstractThe 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.
Marco A. Saraiva and M. Helena Florêncio
Elsevier BV
Marco A. Saraiva and M. Helena Florêncio
Elsevier BV
Marco A. Saraiva and Maria Helena Florêncio
MDPI AG
Parkinson’s disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder. The aggregation of the amyloid disordered protein α-synuclein (Syn) has been implicated in the development of PD. In the literature, it has been suggested that tyrosine residues of Syn play an important role in the interactions established during the fibrillation process. Herein, the prevalence of the referred interactions under shear stress conditions of Nα-acetyl-L-tyrosinamide (NAYA) and of Syn solutions by using membrane centrifugal filters with different cut-off of 200 nm, 100 kDa, 50 kDa and 30 kDa, under centrifugation conditions, were investigated. In order to determine the nature of the interactions involving the protein tyrosine residues the NAYA compound, which mimics the peptide bonds in protein and also possesses a tyrosyl group similar to the tyrosyl groups found in the Syn protein molecular structure, was used. It is expected that for a small molecule, such as NAYA, no molecular association occurs, contrary to what exists in the Syn protein solutions, which can more adequately retrieve the type of interactions formed, involving the tyrosyl group. Therefore, sensing the tyrosyl group absorption, spectroscopic techniques, in particular, were used. For NAYA, an intramolecular interaction between the tyrosyl group and the peptide bond was evidenced. For NAYA and Syn, it was observed that decreasing the membrane centrifugal filters pore size, under centrifugation conditions, was concomitant with the minimization of the intramolecular interactions between the tyrosyl group and the peptide bond. With this, it is likely to assume that shear stress conditions in the Syn solutions propel protein aggregation by a less strained protein backbone. Contrary to the centrifugation of NAYA solutions, centrifuging Syn solutions revealed molecular association and a progressive exposure of protein tyrosyl groups to water. Thus, we can also infer that shear stress conditions in the Syn solutions cause the protein tyrosyl groups to not intervene in the protein aggregation.
Marco A. Saraiva and M. Helena Florêncio
Elsevier BV
Marco A. Saraiva
Elsevier BV
Marco A. Saraiva
Elsevier BV
Marco A. Saraiva, Carlos M. Borges, and M. Helena Florêncio
Springer Science and Business Media LLC
Marco A. Saraiva, Carla D. Jorge, Helena Santos, and António L. Maçanita
Elsevier BV
Marco A. Saraiva, Carlos M. Borges, and M. Helena Florěncio
SAGE Publications
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 with regard to 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.
Marco A. Saraiva, Carlos M. Borges, and M. Helena Florêncio
SAGE Publications
Our previous experiments on electrospray ionisation mass spectrometry (ESI-MS) analysis of reaction mixture solutions containing 4-(−2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), a commonly used buffer, indicated that HEPES species did not significantly suppress analyte species, even in reaction mixture solutions with significant amounts of HEPES. With the purpose of investigating the behaviour of HEPES under ESI-MS conditions, HEPES aqueous solutions and HEPES aqueous solutions containing analyte with high- and low polarity and with different acid/base chemistry, were therefore investigated. For electrosprayed aqueous solutions of HEPES with concentrations above 10−5 M, an enhanced formation of HEPES multimer ions, showing HEPES monomer ion formation, was observed. This enhanced formation of HEPES multimer ions is much higher than those observed for other polar compounds, such as acetyl–arginine, acetyl–lysine and histidine. Information from solution behaviour such as HEPES concentration, solution pH and instrumental factors, namely the capillary temperature, was related to information from mass spectra. The results obtained led us to conclude that the formation of HEPES ions is related to the initial solution composition. The influence of analyte species on HEPES species formation, for electrosprayed HEPES solutions with analyte, was also investigated. The variations observed for HEPES monomer and multimer ion abundances, which were found to be consistent with those observed for analyte monomer ion abundances, were related to the type of analyte, i.e. to their acid/base nature. Strikingly, the variations observed between HEPES monomer and multimer ion abundances enable the discrimination of different influences of analyte species on HEPES species formation. The results obtained also provided an explanation for the observation that HEPES species do not significantly suppress analyte species ion signals when highly-concentrated HEPES solutions with analyte are electrosprayed. According to our results, the associated behaviour between HEPES species seems to be preserved in the gas phase during electrospray ionisation. This observation may provide some information that may be useful regarding the behaviour involved in the gas-phase ion formation process from charged droplets during electrospray ionization or, at least, to differentiate between behaviours.
Marco A. Saraiva, Carlos M. Borges, and M. Helena Florêncio
Wiley
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.
Marco A. Saraiva, Carlos M. Borges, and M. Helena Florêncio
Wiley
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.
Marco A. Saraiva, Carlos M. Borges, and M. Helena Florêncio
Wiley
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.