Journal of Molecular Biology
Volume 386, Issue 3, 27 February 2009, Pages 878-890
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Competition between Folding, Native-State Dimerisation and Amyloid Aggregation in β-Lactoglobulin

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Abstract

We show that a series of peptides corresponding to individual β-strands in native β-lactoglobulin readily form amyloid aggregates and that such aggregates are capable of seeding fibril formation by a full-length form of β-lactoglobulin in which the disulfide bonds are reduced. By contrast, preformed fibrils corresponding to only one of the β-strands that we considered, βA, were found to promote fibril formation by a full-length form of β-lactoglobulin in which the disulfide bonds are intact. These results indicate that regions of high intrinsic aggregation propensity do not give rise to aggregation unless at least partial unfolding takes place. Furthermore, we found that the high aggregation propensity of one of the edge strands, βI, promotes dimerisation of the native structure rather than misfolding and aggregation since the structure of βI is stabilised by the presence of a disulfide bond. These findings demonstrate that the interactions that promote folding and native-state oligomerisation can also result in high intrinsic amyloidogenicity. However, we show that the presence of the remainder of the sequence dramatically reduces the net overall aggregation propensity by negative design principles that we suggest are very common in biological systems as a result of evolutionary processes.

Introduction

Despite the natural tendency of proteins to fold into native structures unique to their amino acid sequences,1, 2 they are capable of aggregating both in vitro and in vivo into large assemblies with a variety of morphological features.3, 4, 5, 6 Of such aggregates, amyloid fibrils represent particularly well-ordered structures that can be micrometers in length while just a few nanometers in diameter and contain cross β-strands aligned perpendicularly to the fibril axis.7, 8, 9, 10, 11 Such fibrils are the subject of intense interest at the present time since their deposition in tissue is associated with a range of increasingly common human diseases, including both systemic and organ-specific conditions, such as Alzheimer's disease and type II diabetes.4, 12, 13 Remarkably, many peptides and proteins, and indeed homopolymers, such as polyalanine and polythreonine, without any known involvement in amyloid disease can also form amyloid fibrils in vitro,14, 15, 16, 17, 18, 19, 20, 21 suggesting that the ability to form amyloid fibrils is an intrinsic property of polypeptide chains.22 Furthermore, such structures have the potential to generate new forms of nanoscale biomaterials based on the amyloid fibrillar architecture since they are characterised by well-ordered and stable structures that can be formed by self-association in aqueous solution.23, 24, 25, 26 Thus, elucidation of the mechanism by which polypeptide chains convert into amyloid fibrils is an important goal in medical, biological and material sciences.

Formation of amyloid fibrils appears to occur in a nucleation-dependent manner that is followed by a rapid extension process that gives rise to fibrillar architectures.27 It has been observed by analysis of native-state structures that the existence of exposed β-strands at the edge of β-sheet structures tends to be avoided since they help in the formation of β-aggregates, such as amyloid fibrils.28 Extensive surveys of structures in the Protein Data Bank suggest that a variety of mechanisms prevent the exposure of hydrophobic surfaces in such edge strands (e.g., by formation of intermolecular β-sheets or by placing inward-pointing charged side chains).28

One of the proteins exemplifying the former situation is bovine β-lactoglobulin, which has been shown to be capable of forming amyloid fibrils by prolonged incubation at pH 7.0 in the presence of 5.0 M urea.18 However, the reaction is relatively slow and can take several weeks to reach completion. β-Lactoglobulin forms a homodimeric native state at neutral pH in the absence of denaturant, although it dissociates into monomeric but still natively folded species at pH values below 3.0.29 In the native state, the protein has a predominantly β-sheet structure, consisting of a β-barrel of eight antiparallel β-strands (A–H) and an additional edge β-strand (I) that is part of the dimer interface located at the edge of the β-sheet of each individual molecule, although there is one major α-helix at the C-terminus (Fig. 1). The β-barrel structure has the shape of a flattened cone resembling a calyx involving the N- and C-terminal β-sheets; the N-terminal β-sheet consists of the βB, βC and βD strands, while the C-terminal one involves the βA, βE, βF, βG, βH and βI strands. The protein also contains two disulfide bonds (C66–C160 and C106–C119, linking strands βD–βI and βG–βH, respectively) and one free cysteine residue (C121). Despite the fact that the native structure of β-lactoglobulin contains a predominantly β-sheet structure, the sequence of β-lactoglobulin encodes a relatively high α-helical propensity; indeed, the major folding intermediate observed to be populated during the folding of the protein contains non-native α-helical motifs30, 31, 32, 33, 34, 35, 36, 37 located mainly in the vicinity of the βA-strand region in the native structure.35

In this work, we describe studies on the relative abilities of peptide fragments corresponding to the amino acid sequences of the β-strands and the α-helical regions of the hydrophobic core of native β-lactoglobulin to form amyloid fibrils and to seed the aggregation of full-length β-lactoglobulin. These results reveal new insights into the general principles underlying the competition between folding, native-state oligomerisation and multimolecular aggregation and provides specific information about the way these factors influence the behaviour of β-lactoglobulin itself.

Section snippets

Amyloid formation by peptide fragments of β-lactoglobulin

The peptide fragments that we have analysed here correspond to the βA, βF, βG, βH and βI strands and the C-terminal α-helical region (α1) of native β-lactoglobulin (Fig. 1); the amino acid sequences are shown in Fig. 1b. Except for βA, the peptides are all located at the C-terminal β-sheet or at the dimer interface of the native protein. These fragments were chosen because the corresponding regions of the sequence form the hydrophobic core of the native structure of the full-length protein.36

Materials

The A variant of bovine β-lactoglobulin, which differs from the B variant at positions 64 and 118, where an aspartate and a valine in the A variant are substituted by a glycine and an alanine in the B variant, was purchased from Sigma (St. Louis, MO). RCM-β-lactoglobulin was prepared as described in Ref. 32. Other chemicals were purchased either from Nacalai Tesque (Kyoto, Japan) or from Wako Pure Chemical Industries (Osaka, Japan). The polyvinyl formal–carbon-coated 200-mesh copper grids

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (no. 15076201) and the Global COE Program A08 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to D.H.) and through grants from the Leverhulme and Wellcome Trusts (to M.V and C. M. D.). D.H. acknowledges Dr. Takashi Fukui for assistance in CDpro analysis. A.P. acknowledges with gratitude the receipt of a Cambridge Gates Scholarship; M.V., that of a Royal Society University

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