Macromolecular crowding modulates the kinetics and morphology of amyloid self-assembly by β-lactoglobulin

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Abstract

The formation of amyloid fibrils by β-lactoglobulin in the presence of GndHCl has been monitored by using thioflavin T (ThT) fluorescence, Congo Red and transmission electron microscopy (TEM). Large quantities of aggregated protein are formed by incubating β-lactoglobulin in 2 M GndHCl at room temperature and pH 7.0 for about 20 days. The kinetics of fibrillation process can be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). Moreover, the effects of macromolecular crowding agents, Dextran 70 and polyethylene glycols (PEG), on the amyloid formation of β-lactoglobulin at pH 7.0 are studied. The results show that the increase in macromolecular crowding agent concentrations results in shorter lag time and faster growth of fibrils. It proves that macromolecular crowding can effectively accelerate the fibril formation of β-lactoglobulin at neutral pH. At the same time, it can be observed that the amplitude of the ThT fluorescence intensity decreases as the Dextran 70 concentration is increased. The observation suggests that the yield of amyloid fibrils is significantly reduced by the addition of macromolecular crowding agents. The conclusion is further confirmed by the transmission electron microscopy. In addition, the results of transmission electron microscopy also indicate that macromolecular crowding can alter the fibril morphology of β-lactoglobulin. In brief, our findings demonstrate that the effects of macromolecular crowding are essential to the understanding of protein amyloid self-assembly occurring in vivo.

Introduction

The association of partially unfolded or completely unfolded proteins or peptides results in the amyloid fibrils [1], [2], [3]. The formation of amyloid fibrils is of intense medical interest at the present time since the deposition of the amyloid in vivo can cause numerous serious diseases, including Alzheimer's disease, prion encephalopathies, Huntington's disease and type II diabetes [4], [5], [6], [7], [8], [9]. So far, about 30 different proteins have been found to be involved in these amyloidoses. Although the proteins involved in diseases have different sequences and tertiary structures, they can all form the amyloid fibrils with the same morphological characteristics [10]. Remarkably, it has been demonstrated that most proteins or peptides not associated with diseases can also form the amyloid fibrils in designed conditions, suggesting that the ability to form the amyloid fibrils is a common property of all proteins or polypeptide chains [11], [12], [13].

Bovine β-lactoglobulin is a dimeric protein at neutral pH and dissociates into monomers but it is still natively folded species at pH values below 3.0 [14], [15], [16]. It is composed of 162 amino acid residues with an 18.4 kDa molecular weight. The conformation of the protein at both neutral and acidic pH has been determined by X-ray crystallography and NMR spectroscopy. It is a predominantly β-sheet protein consisting of nine β-strands (A–I) and one major helix at the C-terminal end of the molecules [17]. The protein also contains two disulfide bonds and one free cysteine residue. It has been shown that β-lactoglobulin at pH 7.0 can form fibrils in vitro under appropriate conditions, in which the protein is likely to be at least partially unfolded, for example, at higher temperature or in the presence of urea [18], [19], [20], [21].

Amyloid formation has been generally studied in dilute solutions [22], [23]. However, cellular medium is crowded since there are many different species of macromolecules within a cell, including proteins, nucleic acids, lipids and carbohydrates [24], [25]. Those intracellular solutes present in the cell occupy a significant part of the total cell volume. Since steric repulsion exists between macromolecules, the accessible volume of single molecule is reduced. Such effect is called excluded volume, which may result in significant alterations of biology processes. This means that the biochemical processes in vivo are different from those in vitro, so it is of paramount importance to study the effects of macromolecular crowding in order to understand the living processes in vivo. Firstly, Minton and Zimmerman studied the effects of macromolecular crowding on the biological processes and established the field of molecular crowding [26]. After that, several groups have also discussed the effects of macromolecular crowding on various biological processes including protein native structure, stability, protein folding, binding and aggregation [27], [28], [29], [30]. Specifically, there have been several systematic investigations of the macromolecular crowding effects on the amyloid self-assembly of proteins [31], [32]. The results demonstrated that macromolecular crowding can guide protein aggregation and elevate the amount of aggregated peptide.

In this study, the effects of macromolecular crowding on amyloid formation of β-lactoglobulin at neutral pH were investigated by using ThT, Congo Red and transmission electron microscopy. The findings demonstrate that macromolecular crowding can significantly accelerate the amyloid formation of β-lactoglobulin. In addition, it is shown that macromolecular crowding can decrease the amount of amyloid fibrils and alter the fibril morphology of β-lactoglobulin.

Section snippets

Chemicals

Bovine β-lactoglobulin was obtained from Sigma (Sigma–Aldrich Co., St. Louis, MO) and was used without purification. Dextran 70, PEG (with molecular weights of 400, 8000 and 20,000), Thioflavin T (ThT), sodium azide (NaN3) and Congo Red (CR) were purchased from Sigma. All other chemical regents used were of analytical grade and were made in China.

Tryptophan fluorescence measurements

A stock solution of β-lactoglobulin was prepared in 20 mM PBS buffer at pH 7.0. β-Lactoglobulin solutions of 0.05 mg/ml concentration were used for

Fibril formation in GndHCl solutions

The increased fluorescence emission of the ThT is usually used for monitoring the kinetics of amyloid fibril formation, which is a specific marker for the cross β-sheet conformation of amyloid structures [33]. The β-lactoglobulin solutions containing 2 M GndHCl were incubated at room temperature and pH 7.0. ThT fluorescence intensity for β-lactoglobulin in 2 M GndHCl is shown as a function of time in Fig. 1.

When the BLG solutions were incubated in the presence of 2 M GndHCl, a dramatic increase in

Discussion

More and more experimental results suggest that if a given polypeptide or protein is incubated under appropriate conditions, it should be possible to promote amyloid self-assembly of proteins. Generally, such conditions are likely to be that the proteins are of partially unfolded state. The present results for β-lactoglobulin support this idea and provide further evidence to prove that partial unfolding for proteins is a prerequisite to amyloid fibril formation.

Many experiments concerning the

Conclusion

In summary, the effects of macromolecular crowding on the fibril kinetics of β-lactoglobulin were systematically studied by the fluorescence probe, ThT, and shown to be consistent with the increase of effective β-lactoglobulin concentration, indicating that the excluded volume effect plays an important role. Additionally, these results also demonstrate that macromolecular crowding can diminish the fibril yield and change the fibril morphology of β-lactoglobulin.

Acknowledgements

This work is supported by the Natural Science Foundation of China (no. 11171155) and by the Fundamental Research Funds for the Central Universities (no. Y0201100265). Baoliang Ma was supported by the Youth Foundation of Nanjing Agricultural University (05060005).

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