Journal of Molecular Biology
Competition between Folding, Native-State Dimerisation and Amyloid Aggregation in β-Lactoglobulin
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
References (63)
Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world
Biochim. Biophys. Acta
(2004)Protein misfolding, evolution and disease
Trends Biochem. Sci.
(1999)- et al.
Engineering amyloidogenicity towards the development of nanofibrillar materials
Trends Biotechnol.
(2004) - et al.
Designing supramolecular protein assemblies
Curr. Opin. Struct. Biol.
(2002) - et al.
The equilibrium intermediate of β-lactoglobulin with non-native α-helical structure
J. Mol. Biol.
(1997) - et al.
The burst-phase intermediate in the refolding of β-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy
J. Mol. Biol.
(1996) - et al.
Is folding of β-lactoglobulin non-hierarchic? Intermediate with native-like β-sheet and non-native α-helix
J. Mol. Biol.
(2000) - et al.
Trifluoroethanol-induced stabilization of the α-helical structure of β-lactoglobulin: implication for non-hierarchical protein folding
J. Mol. Biol.
(1995) - et al.
The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner
J. Mol. Biol.
(2006) - et al.
Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the β-domain
J. Mol. Biol.
(2000)
Identification of minimal peptide sequences in the (8–20) domain of human islet amyloid polypeptide involved in fibrillogenesis
J. Struct. Biol.
Investigation of a peptide responsible for amyloid fibril formation of β2-microglobulin by Achromobacter protease I
J. Biol. Chem.
Amyloid-forming peptides from β2-microglobulin—insights into the mechanism of fibril formation in vivo
J. Mol. Biol.
Ultrastructural organization of amyloid fibrils by atomic force microscopy
Biophys. J.
Dependence on solution conditions of aggregation and amyloid formation by SH3 domain
J. Mol. Biol.
Prediction of “aggregation-prone” and “aggregation-susceptible” regions in proteins associated with neurodegenerative diseases
J. Mol. Biol.
Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae
Cell
Identification of the core structure of lysozyme amyloid fibrils by proteolysis
J. Mol. Biol.
Prediction of aggregation-prone regions in structured proteins
J. Mol. Biol.
Native-like β-hairpin retained in the cold-denatured state of bovine β-lactoglobulin
J. Mol. Biol.
β-Lactoglobulin assembles into amyloid through sequential aggregated intermediates
J. Mol. Biol.
Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set
Anal. Biochem.
Probing the origins, diagnosis and treatment of amyloid diseases using antibodies
Biochimie
Calculation of protein extinction coefficients from amino acid sequence data
Anal. Biochem.
Prediction of local structural stabilities of proteins from their amino acid sequences
Structure
Protein folding: a perspective from theory and experiment
Angew. Chem.
Principles that govern the folding of protein chains
Science
Protein folding and misfolding
Nature
Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution
J. Mol. Med.
Towards complete descriptions of the free-energy landscapes of proteins
Philos. Trans. R. Soc. London, Ser. A
High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy
Proc. Natl Acad. Sci. USA
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