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Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain1

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Abstract

The formation of amyloid fibrils by the SH3 domain of the α-subunit of bovine phosphatidylinositol-3′-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in β-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis.

Introduction

Amyloid fibrils are proteinaceous deposits that are associated with a range of human disorders including Alzheimer’s disease and the transmissible spongiform encephalopathies. 1 There is growing evidence that partial or complete unfolding of the specific protein associated with each disease is the first step in the conversion of an otherwise soluble protein into these aggregated structures. 2, 3, 4 Despite the different characteristics of the precursor proteins, all amyloid fibrils are long and unbranched and display a common core cross-β structure 5 suggesting that amyloid formation by the different precursors must share common features. Although considerable advances are being made in the structural characterisation of amyloid fibrils and the underlying mechanism of their formation, 4 many aspects of the process of fibril formation are still not clear. Recently, a range of proteins not related to any human disease have been found to form fibrils in vitro under mildly denaturing conditions. 6, 7, 8, 9, 10 This observation has led to the suggestion that the ability to form amyloid fibrils is a common phenomenon and a generic property of polypeptide chains. 4, 6, 11 The ability to form amyloid fibrils from a wide range of proteins gives access to a large number of model systems with which to study the process of fibril formation in more detail.

One particularly favourable system for investigation of amyloid fibril formation is the SH3 domain of the α-subunit of bovine phosphatidylinositol-3′-kinase (PI3-SH3). Not only is this a protein with a very well defined globular structure, but there is considerable information concerning the structure of the amyloid fibrils that this domain forms readily at acidic pH values. 12, 13 PI3-SH3 is a β-sheet protein 14 that forms a relatively compact denatured state in solution at low pH. 6 The protein slowly aggregates from this state into long fibrils with characteristics closely similar to those of amyloid fibrils associated with human disease. The highly ordered structure of one of the morphologies exhibited by these fibrils has permitted a low-resolution electron density map to be determined by novel cryo-electron microscopy (cryo-EM) techniques. 12 A model of the fibrils constructed to be consistent with this map and with X-ray fibre diffraction data contains four protofilaments, each of which is composed of a pair of β-sheets that is only very slightly twisted around the fibril axis. This map also confirms that the native protein must unfold substantially prior to the assembly of the polypeptide chain into the fundamentally different structure present in the fibrils. 12 Additional studies by EM and atomic force microscopy (AFM) indicate that the protofilaments are wound around each other in a left-handed manner. 13

Despite extensive studies of amyloid fibrils that have resulted in the elucidation of many aspects of their underlying nature, important issues concerning their structure and mechanism of formation still remain to be established. Little is known about the properties of the polypeptide chains in solution prior to assembly into amyloid fibrils or about the molecular details of the conformation of the polypeptide chain within the fibrils. To approach such questions, a detailed analysis of the pH-denaturation profile of PI3-SH3 has been performed in order to link the properties of the soluble precursors with those exhibited by the various aggregated states formed by the protein under different conditions. The results reveal that subtle differences in solution conditions can influence dramatically the ability to form organised fibrils relative to amorphous material, as well as the detailed morphology of the fibrillar structures obtained.

Section snippets

Results

Native PI3-SH3 has been denatured at low pH to study the relationship between the characteristics of the soluble protein, its aggregation kinetics and the nature and degree of organisation of the aggregates it forms, using a variety of biophysical techniques.

Precursor compactness and protein aggregation

The present results indicate that the nature of the aggregates formed by PI3-SH3 is highly dependent on the pH of the solution in which aggregation takes place. NMR spectra of the protein demonstrate that the native structure of the protein is progressively lost at pH values below 4.0 as the protein unfolds. NMR pulse-field gradient experiments show that the effective hydrodynamic radius of the protein increases on unfolding but to a value that is significantly smaller than that of the protein

Conclusions

The results described here confirm that it is necessary to unfold the PI3-SH3 domain in order for it to form stable and ordered aggregates, and ultimately well-defined amyloid fibrils. This conclusion is consistent with existing observations in a range of systems and is indicative of the need for the native structure of soluble proteins to be disrupted to allow the polypeptide chain to associate into β-sheet structures characteristic of amyloid aggregates. 29, 35, 53, 54 Aggregation of the

Protein expression and purification

PI3-SH3 was expressed in Escherichia coli BL21 strain, and purified as described. 57 The protein was also expressed in E. coli BL21 (DE3) strain as a fusion with a (histidine)6-tag at the N terminus using the vector pET14b (Novagen, Madison, WI). The fusion protein was passed through a metal-chelate resin (Poros, Applied Biosystems, Foster City, CA) pre-loaded with Ni2+, and eluted with a gradient of 5 mM–250 mM imidazole in 50 mM sodium phosphate buffer (pH 7.0), 0.5 M NaCl. The eluted protein

Acknowledgements

We thank Dr Deborah Wilkins and Dr Jonathan Jones for assistance with the NMR diffusion measurements, and Dr Mario Bouchard for helpful discussions. J.Z. was supported by grants from the European Commission (EC) and the Wellcome Trust. I.G. acknowledges a grant from the EC. J.L.J. was supported by a grant of the Wellcome Trust. The research of C.M.D. is supported, in part, by a programme grant from the Wellcome Trust. The Oxford Centre for Molecular Sciences is supported by the UK BBSRC, MRC

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    Edited by P. E. Wright

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    Present addresses: J. L. Jiménez, Computational Genome Analysis Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK; Christopher M. Dobson, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.

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