Interpretation of α-synuclein UV absorption spectra in the ...

Highlights

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  Early large α-synuclein aggregates populate the solutions.

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  Interpretation of α-synuclein UV absorption spectra.

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  Label-free analysis of α-synuclein aggregation.

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  Stabilization of α-synuclein by intermolecular contacts involving tyrosine residues.

Abstract

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.

Introduction

The misfolding and aggregation of otherwise soluble proteins into fibrillar amyloid deposits is linked with several pathogeneses which include approximately 50 human neurological and systemic disorders [1]. Although the formation of fibrillar amyloid deposits in the human body is linked with disease, the mentioned disorders include a number of disparate amounts of symptoms while some of these are among the most common human debilitating disorders such as Alzheimer's disease and Parkinson's disease (PD) [1]. Recently, there is growing evidence that the formation of oligomeric species, formed in the early stages of the amyloid fibrils, are more cytotoxic than the amyloid fibrils themselves [1]. Recent advances on the mechanism underlying the oligomeric species formation have indicated that the high content of solvent-exposed hydrophobic regions in the biological molecules contributed to their association [1]. Therefore, it is crucial to unravel the early stages of amyloid fibrils formation which includes the conservative oligomeric species formation [[2], [3], [4], [5], [6], [7], [8]]. Numerous attempts have been made in the identification and characterization of such oligomeric species but due to their solubility, transient and highly heterogeneous nature, difficulties arise in the determination of such intermediate species at the molecular level and disparate results can be encountered in the literature [1]. Concerning the early stages of Syn aggregation, a recent study revealed the type of oligomeric species involved in the so-called stepwise aggregation mechanism [9]. The protein α-synuclein (Syn), which is an almost complete disordered small protein at physiological pH, has been thoroughly studied due its relation with PD, in particular. This protein has a natural ability to aggregate, and brain inclusions rich with this protein have been found in patients suffering from PD. Structurally, Syn with a molecular mass of 14,460 Da comprising 140 residues and possess three regions. The regions are the N-terminal region with amphipathic helices containing a conserved 6 repetitive amino acids motif KTKEGV (residues 1–60), an amyloidogenic NAC (non-amyloid component) region (residues 61–95) and a C-terminal acidic tail region (residues 96–140). In a previous report, since Syn possesses four tyrosine residues and no tryptophan, a detailed study of the molecular fluorescence emission characteristics of these four tyrosine residues was undertaken in the course of the early stages of the aggregation process of Syn [10]. Herein, I have conducted a detailed study of the UV absorption spectra of this protein in the peptide bond (200–230 nm) and the aromatic regions for several protein concentrations (16.7, 33.5, 50.2, 67.0 and 83.7 μM), several solution pH (7, 3 and 2) and during the first 300 min of reaction. Due to the fact that Syn hydrophobic collapse occurs at low pH, it was found convenient not only to study Syn aggregation at neutral pH but also at low pH. In literature, none of these regions (peptide bond and aromatic regions) have been studied in detail for this protein, i.e. in what the UV absorption spectra are concerned. The UV absorption of proteins in the range 180 to 230 nm is due almost entirely to π → π∗ transitions in the peptide bonds. Absorption in the range of 230–300 nm is dominated by the aromatic side-chains of tryptophan, tyrosine, and phenylalanine residues, and there is weak contribution by disulphide bonds near 260 nm. Crammer and Neuberger (1943) [11] and Shugar (1952) [12] were the first to use UV–Vis spectroscopy to study the optical properties of the tyrosine chromophore and to interpret changes in absorbance spectra of proteins as a function of pH. Furthermore, the UV absorption spectra of polypeptides and proteins were thoroughly investigated during the decades of 50 and 60 of the last century [[13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35]]. It was convenient in that time studying polypeptides and globular proteins that adopt different conformations in their folded (e.g. α-helix conformation) and unfolded states (random coil conformation) and following this transition state with UV absorption spectroscopy, optical rotatory dispersion (ORD) and circular dichroism. However, much less is known concerning the scenario in proteins like Syn lacking compact secondary and tertiary structures. Nevertheless, for polymers of amino acids, such poly-L-aspartic and poly-L-glutamic acids, it has been reported in the 60's that the peptide bond molar extinction coefficient of both α-helices and random coil conformations are influenced by the counter-ion concentration in solution, especially in the random coil conformation [34]. More particularly, the molar extinction coefficient of the peptide bond in the random coil of such polymers increased when decreasing the counter-ion concentration in the polymers solutions [34]. This result can be particularly interesting in the case of the Syn disordered protein and was thus here explored. I found out that at solution pH 3 the protein Syn is more compact than at solution pH 2, and at both mentioned solutions pH the protein Syn is being collapsed. It was also observed that for a 33.5 μM protein solution concentration, in particular, not only the counter-ion concentration increases from solution pH 7 to 3 and to 2, as expected, but also data from UV absorption spectra at this referred protein solution concentration can be used quantitatively to monitor the progression of the Syn aggregation reaction in the lag-phase, particular. With regard to the aromatic region in the UV absorption spectra of Syn, a band with a maximum at ca. 293 nm was determined in the UV difference spectra pH 7 – pH 3 and pH 7 – pH 2 of Syn which is not related with the aggregation of the protein at pH 3 and 2 in comparison to pH 7 but it is with the formation of intermolecular hydrogen-bonded complexes of tyrosine residues with aspartic and glutamic acids residues.

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Section snippets

Materials

All chemicals, including Nα-acetyl-L-tyrosinamide (NAYA), were of analytical grade and obtained from Sigma-Aldrich.

α-Synuclein Expression and Purification

The pT7–7 plasmid containing the human SYN sequence (kindly provided by Professor Doctor T. Outeiro, IMM, University of Lisbon) was used to overexpress Syn in Escherichia coli BL21 bacteria. Syn was purified as previously described [10]. Briefly, Syn was purified by boiling the cell homogenate, which precipitates most cellular proteins, leaving Syn as the major component of the

Identification and Characterization of Syn Solutions Species by Dynamic Light Scattering

Fig. 1a presents the size distributions represented as intensity (normalized) for a 33.5 μM protein solution concentration at pH 7, 3 and 2 (10 mM Tris-HCl) (20 °C). The DLS measurements were obtained 2–5 min after dilution to a 33.5 μM protein concentration of a more concentrated protein stock. The protein stock solution was previously centrifuged with 100 kDa cut-off filters in order to remove possible existent high molecular weight aggregates. All materials were properly cleaned and the

Conclusions

By varying the protein concentration, solution pH and time of aggregation, the UV absorption spectra of Syn were studied in detail with respect to the peptide bond (200–230 nm) and the aromatic regions during the first 300 min of reaction. It is the first time that those regions in the UV absorption spectra of Syn were studied thoroughly. In what the peptide bond region is concerned, it was determined that, Syn solutions at pH 3 and 2, although Syn is being collapsed, the protein is slightly

Declaration of Competing Interest

The author declares that there is no conflict of interest.

Acknowledgements

I acknowledge Professor António L. Maçanita (IST, UL) for helpful discussions and Professor Helena Santos lab (ITQB, UNL) for the expression and purification of the amyloid protein Syn. M. A. Saraiva also acknowledges the FCT for the post-doc grant SFRH/BPD/37016/2007.

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