Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer’s Aβ peptide

https://doi.org/10.1016/j.bbrc.2011.04.141Get rights and content

Abstract

The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer’s disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1–40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.

Highlights

► Sodium phosphate buffer accelerated Aβ(1–40) nucleation relative to HEPES. ► Aβ(1–40) fibrils formed in the two buffers show only minor structural differences. ► NMR revealed that Aβ(1–40) histidine residues mediate buffer dependent changes.

Introduction

Amyloid fibrils are a predominant biochemical feature of several degenerative diseases, including Alzheimer’s disease [1]. Different environmental factors are known to influence this reaction, including pH value, temperature, salts and other cosolutes [2], [3], [4], [5], [6]. However, though various factors which influence the structural properties of the Alzheimer’s disease-associated amyloid-forming peptides have been identified, the specific effects of phosphate buffer have yet to be established. Whilst previous investigations have observed differences in Aβ fibrils formed under different buffer conditions, such studies simultaneously varied additional parameters, such as pH value and temperature, which are known to affect Aβ aggregation themselves [2], [3], [4], [5], [6].

To analyze the specific influence of phosphate buffer on Aβ(1–40) fibrillation and oligomerization, we have examined this mechanism in the presence of sodium phosphate as compared to the use of 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES). We used a battery of techniques to examine the effects of phosphate and HEPES buffers on the peptide solution structure, fibrillation kinetics and fibril structure of Aβ(1–40). Our results reveal that significant, and histidine-dependent, changes occur in the fibrillation and oligomerization pathways of Aβ(1–40) directly due to the buffer system employed.

Section snippets

Expression of Aβ peptide

Aβ(1–40) and 15N-labeled Aβ(1–40) were prepared by in house recombinant expression according to published procedures, as described previously [7], [8], [9] using a 10 l Biostat ED10 stirred bioreactor. The composition of the phosphate salts in the medium were modified using 1.5 g/L potassium dihydrogen phosphate and 0.975 g/L disodium hydrogen phosphate, dodecahydrate. For biosynthetic 15N-labeling, 15N-labeled ammonium chloride (Cambridge Isotope Laboratories, Inc.) was used as the sole nitrogen

Results

When 50 μM peptide is incubated in 50 mM of either phosphate or HEPES buffer at pH 7.4 (Fig. 1A and B), we observe significant differences in the measured fibrillation kinetics (Fig. 1C). The lag time tl, which relates to the length of the rate limiting step of nucleus formation [15], [16], was determined according to the methods outlined previously [8]. We observed a reduced tl of 15.9 ± 1.2 h in sodium phosphate, compared to 20.4 ± 2.2 h in HEPES. A smaller tl value is also recorded with phosphate

Discussion

All current data signify that phosphate and HEPES buffers present different and specific interactions with the His side chains of Aβ. The specificity of the buffer-dependent effects concerning His are very interesting, as these side chains were previously identified to present strong interactions between Aβ with Cu2+ and other metal ions, potentially mediating metal-dependent changes of Aβ aggregation and fibrillation [26], [27]. His residues are known to be of importance to protein stability

Acknowledgments

This work was supported by BMBF (BioFuture), DFG (SFB 610), ProNet-T3 (BMBF), the country Sachsen-Anhalt and the European Regional Development Fund (ERDF). We thank M. Kirchner and K. Pflüger for technical assistance.

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