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Images of prion fibrils and fibers
Universal architecture for amyloid diseases
N-terminal conformational change inferred
Misfolding in GSS
Hydrophobic patches at low pH

Prion fibrils and fibers

More images are posted in the Gallery. A universal cross-beta structure describes the congophilic fiber of amyloid diseases, including prion disease, as noted by GG Glenner in 1980, NEJM 302:1283;1333. Chou and Martin determined the dimensions of prion fibrils in 1971. The amyloid fiber is a coil of several fibrils, themselves helical beta sheet of strands perpendicular to a cylindrical axis. The repeat unit is 115 angstroms; a peptide strand occupies 4.8A; so 24 strands per repeat with rotation of 360/24=15 degrees and right-hand twist [Chothia 1973].

The gifs show 12 fibrils (groupable as 1, 2, 3, 4, or 6) wrapping with a hollow (resp. solid) tube of ratio 44:100 and a non-supercoiled fiber comprised of 4 fibrils (roughly , transthyretin). Perhaps 3-4 consecutive strands on a sheet are grouped as prion monomer, the next 3-4 as a second monomer oriented so the junction is anti-parallel (and binds Congo Red).Interactive parallel and anti-parallel beta sheets from PDB show hydrogen bonding and side-chain positioning. More difficult virtual reality files enable you to fly-throughs of the prion fibril.

Common Core Structure of Amyloid Fibrils by Synchrotron X-ray Diffraction.

Sunde M, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CCF
J Mol Biol 1997 Oct 31;273(3):729-739  Oxford fulltext free as pdf
Tissue deposition of normally soluble proteins as insoluble amyloid fibrils is associated with serious diseases including the systemic amyloidoses, maturity onset diabetes, Alzheimer's disease and transmissible spongiform encephalopathy. Although the precursor proteins in different diseases do not share sequence homology or related native structure, the morphology and properties of all amyloid fibrils are remarkably similar. Using intense synchrotron sources we observed that six different ex vivo amyloid fibrils and two synthetic fibril preparations all gave similar high-resolution X-ray fibre diffraction patterns, consistent with a helical array of beta-sheets parallel to the fibre long axis, with the strands perpendicular to this axis. This confirms that amyloid fibrils comprise a structural superfamily and share a common protofilament substructure, irrespective of the nature of their precursor proteins.


Amyloidosis is the extracellular deposition of insoluble protein fibrils leading to tissue damage and disease (Pepys, 1996; Tan et al., 1995; Kelly, 1996). The fibrils form when normally soluble proteins and peptides self-associate in an abnormal manner (Kelly, 1997). Amyloid is associated with serious diseases including systemic amyloidosis, Alzheimer's disease, maturity onset diabetes, and the prion-related transmissible spongiform encephalopathies (Table 1). There is no specific treatment for amyloid deposition and these diseases are usually fatal. The subunits of amyloid fibrils may be wild-type, variant or truncated proteins, and similar fibrils can be formed in vitro from oligopeptides and denatured proteins (Bradbury et al., 1960; Filshie et al., 1964; Burke & Rougvie, 1972).

The nature of the polypeptide component of the fibrils defines the character of the amyloidosis. Despite large differences in the size, native structure and function of amyloidogenic proteins, all amyloid fibrils are of indeterminate length, unbranched, 70 to 120 A in diameter, and display pathognomonic green birefringence when viewed in polarized light after staining with Congo Red (Pepys, 1996). Early X-ray diffraction examinations of amyloid fibrils (Bonar et al., 1967; Eanes & Glenner, 1968) gave simple patterns with 4.7 to 4.8 A meridional reflections and 10 A equatorial reflections, arising from the molecular spacings present within the regularly repeating, ordered structural elements of the fibrils.

They are characteristic of a cross-b structure (Pauling & Corey, 1951) in which the polypeptide chain is organized in b-sheets arranged parallel to the fibril axis with their constituent b-strands perpendicular to the fibril axis. This distinctive fibre diffraction pattern led to the amyloidoses being called the b-fibrilloses (Glenner, 1980a,b), and the fibril protein of Alzheimer's disease was named the b-protein before its secondary structure was known (Glenner & Wong, 1984). The characteristic cross-b diffraction pattern, together with the fibril appearance and tinctorial properties are now the accepted diagnostic hallmarks of amyloid, and suggest that the fibrils, although formed from quite different protein precursors, share a degree of structural similarity.

In order to determine the extent and nature of this similarity we have used intense synchrotron X-ray beams to obtain the first high-resolution diffraction patterns from a range of different ex vivo and synthetic amyloid fibrils. Amyloid fibrils were isolated from patients with, respectively: monoclonal l immunoglobulin light chain amyloidosis; reactive systemic amyloid A protein amyloidosis and hereditary amyloidosis caused by Leu60Arg variant apolipoprotein A-I ; Asp67His variant lysozyme; and two different transthyretin variants, Val30Met and Gly47Val.

Synthetic fibrils were prepared from a peptide corresponding to residues 10 to 19 (b-strand A) of transthyretin, and from a peptide with the sequence of residues 20 to 29 of the islet-associated polypeptide (IAPP). The high-resolution meridional X-ray pattern and a common repeat on the fibril axis

The synchrotron X-ray diffraction patterns from these different fibril preparations are shown in Figure 1 and the spacings of the reflections are listed in Table 2. These high-resolution patterns are dominated by the cross-b reflections but they also contain groups of additional reflections that have not been observed previously in other amyloid diffraction patterns. Despite the known, large differences in the lengths and folding conformations of the polypeptide chains of the precursors, the major features of the diffraction patterns from the various amyloid fibrils are clearly very similar.

The meridional diffraction pattern derives from the ordered molecular structures along the length of the amyloid fibril. The presence of reflections on the meridian out to 2 A indicates that the individual fibrils have highly ordered internal structures along the fibre axis. The intense reflection at 4.7 to 4.8 A that dominates the meridional diffraction patterns of amyloid fibrils is derived from the mean separation of the hydrogen-bonded b-strands that are arranged perpendicular to the fibre axis in the cross-b structure (Figure 2). In addition to this intense reflection, the synchrotron radiation has also revealed several weaker, higher-angle reflections, occurring in this range of diverse amyloids, that have not been reported previously. These reflections include a second order of the 4.7 to 4.8 spacing, at 2.4 A.

In some of the amyloid samples (Figure 1(b), (d), (e) and (f), the intense 4.7 A reflection can be seen to be a close doublet, with components at 4.82 A and 4.63 A . In patterns where this doublet cannot be resolved its presence can be inferred from the observation that the calculated first order of the second harmonic of the 4.7 A spacing, found at 2.39 to 2.41 A , maps to the extreme inner edge of the intense 4.7 A reflection, leaving space for a 4.6 A component within the overall intensity envelope. The weaker, higher-angle reflections occur at closely similar spacings in the different samples. For example, reflections with spacings of 3.2 A , 2.8 to 2.9 A , 2.22 to 2.27 A and 2.00 to 2.02 A occur frequently, and reflections with spacings of 2.39 to 2.41 A (the second order of the intense 4.82 A reflection) are present in all of the amyloid samples that we have examined at high resolution.

Because these reflections are very weak, even in relatively well oriented patterns, their absence from certain images may simply indicate that they are too weak to be observed above the noise level in those patterns. The observed similarity over the medium- and high-angle regions of the meridional X-ray pattern can only occur if the fibrils have well-defined and closely similar molecular structures, at least insofar as their ordered core components are concerned. The observed meridional spacings can be fitted to the same repeat distance for each pattern, namely 115 A (Table 2). The observed spacings for many of the reflections can also be fitted to a fundamental repeat of 28.8 to 29.9 A to give orders of diffraction of 6 through 14 for spacings from 4.8 A to 2.00 A.

However, this fit does not include all observed reflections. It is possible to include these if it is assumed that the 28.8 to 28.9 A distance represents a pseudo-repeat and that the true repeat is four times as long, being 115.1 to 115.6 A. As Table 2 indicates, all of the observed meridional reflections can be indexed on this longer repeat and this indexing can be carried out separately for the diffraction patterns from each type of amyloid fibril to give almost identical repeat distances. The ability to index the meridional spacings from each different fibril preparation to essentially the same unit cell edge indicates a close similarity in the underlying core molecular structure of all of these samples. This similarity is evidence that the protofilament structure of amyloid fibrils is common across the diverse range of fibril samples examined here, regardless of the constituent protein or the number of protofilaments making up the fibril.

The most intense features of the X-ray patterns we show here correspond to those previously reported for other amyloid fibrils (Table 1), including those from full-length (Kirschner et al., 1986; Gorevic et al., 1987) and fragments of the Alzheimer's disease Ab peptide (Kirschner et al., 1987; Fraser et al., 1991; Inouye et al., 1993), amyloid A protein (Turnell et al., 1986a), calcitonin (Gilchrist & Bradshaw, 1993), insulin (Burke & Rougvie, 1972), and synthetic peptides of transthyretin (Jarvis et al., 1993) and the prion protein (Come et al., 1993; Tagliavini et al., 1993; Nguyen et al., 1995). The lack of high-resolution data, beyond the basic cross-b reflections, has limited further analysis of the molecular structures of these fibrils.

The equatorial X-ray reflections relate to the fibril structure perpendicular to the fibre direction. Because the crystalline order in fibres is usually much lower in directions perpendicular to the fibre axis than parallel to the axis, the equatorial reflection of about 10 A. As the intensities of the equatorial reflections are determined by the structure of the fibrils projected down the fibre axis, this reflection has been identified as representing the spacing of the b-sheets in the amyloid fibril.

The use of synchrotron radiation has revealed a more detailed equatorial diffraction pattern in approximately 10 A, as suggested earlier. It is known that the spacing in b-sheets is dependent on the side-chain composition of the b-sheets (Arnott et al., 1967; Geddes et al., 1968). A recent analysis of the X-ray diffraction patterns from prion rods and fibrils formed from peptides corresponding to fragments of the prion protein has shown that the b-sheet spacings vary in these samples because of differences in sequence (Nguyen et al., 1995). The present results are also in agreement with those of Jarvis and co-workers, who have reported b-sheet spacings of 8 to 10 A in synthetic fibrils prepared from peptides that correspond to single strands of the transthyretin molecule (Jarvis et al., 1993).

The slight variation in the equatorial reflections observed in the series of fibrils presented here therefore presumably reflects the differences in protein sequences in these diverse types of amyloid but falls within acceptable limits for amino acid compositions found in globular proteins. The larger number of equatorial reflections revealed by the synchrotron X-ray source suggests the presence of protofilaments in amyloid fibrils. The short-range order involved in the packing of protofilaments, within or between the fibrils, can be described by a one-dimensional interference function dependent on the centre-to centre separation (or diameter) of the protofilaments, and their arrangement in the fibrils (Burge, 1959, 1963). Electron microscopy studies of the sub-fibrillar structure of amyloid have revealed that different types of amyloid may show different numbers and arrangements of protofilaments in the fibrils (Shirahama & Cohen, 1967; Shirahama et al., 1973; Cohen et al., 1981).

Fraser and co-workers have shown that the protofilaments formed by Ab peptides assemble into hollow rods or ribbons of various sizes under different conditions of pH (Fraser et al., 1991) and, while Ab, amyloid A protein and immunoglobulin light chain amyloid fibrils are reported to be composed of five or six protofilaments around an electron lucent core (Cohen et al., 1981; Kirschner et al., 1987; Fraser et al., 1991), transthyretin amyloid has been shown to be composed of four protofilaments in a square array (Serpell et al., 1995). It may therefore be expected that the equatorial reflections produced by the amyloid fibrils of different types will reflect the variation in number and arrangement of protofilaments in their fibrils.

It is also possible that the diameters of the protofilaments (and therefore their centre-to-centre spacing) may vary because of the need to accommodate differently sized loops linking the b-structure core, and/or to allow for some variation in the lengths of the b-strands dependent on the nature of the precursor. These variations may account for the differences between the 25 to 35 A diameter protofilaments seen in amyloid A protein and immunoglobulin light chain fibrils, and Alzheimer's amyloid and the 50 to 60 A diameter protofilaments demonstrated in transthyretin amyloid (Fraser et al., 1991; Cohen et al., 1981; Serpell et al., 1995).

In view of these possible variations of structure, the observed equatorial spacings listed in Table 3 are difficult to interpret readily in more detail than is given above. Where there is a sufficient number of equatorial reflections and other information it is possible to analyze the substructure of the fibril in some detail, as for example, has been done for the transthyretin fibril (Blake & Serpell, 1996; Blake et al., 1996). The number of observable equatorial reflections listed in Table 3 is insufficient to characterize all of the amyloid fibrils studied here but the similarity in the equatorial reflections displayed by the ex vivo fibrils on the one hand, and the synthetic peptide fibrils on the other, may reflect the fact that the protofilament packing in these two groups is related to the nature of their constituent polypeptides.

A generic fibril structure

The degree of similarity we have observed in the diffraction patterns of these different amyloid samples is indicative of a common core molecular structure at least at the level of the protofilament. The X-ray pattern of one of these fibrils, the Val30Met transthyretin amyloid, has been analysed in detail to generate a novel molecular structure, which has been described (Blake & Serpell, 1996; Blake et al., 1996), and it is reasonable to suppose that its basic structural elements are representative of the other amyloid fibrils examined here. In this molecular model the protofilaments that make up the observed fibrils are composed of a number of b-sheets (four in the case of transthyretin fibrils; but this number may be particular to transthyretin amyloid) running parallel to the axis of the protofilament, with their component b-strands closely perpendicular to this axis.

The regular orientation of the strands and sheets with respect to the fibril axis may account for the magnetic anisotropy observed in amyloid fibril samples, which allows samples of fibrils to be aligned in a magnetic field (Inouye et al., 1993). The diamagnetic anisotropy of the planar peptide bond has the effect that b-sheets, in which the plane of the peptide bond is parallel to the sheet, have a tendency to orient parallel to an applied magnetic field (Worcester, 1978). The meridional reflections from these diverse amyloids are all consistent with the model of the continuous b-sheet helix described in detail for the Val30Met amyloid fibril (Blake & Serpell, 1996). For all of the amyloids, the lowest-order reflection, at 4.8 A , is the 24th order of the 115.5 A repeat, suggesting that the amyloid core contains 24 b-strands in each 115.5 A -long repeating unit along the fibril axis (Figure 3). A twist of the b-sheet through 360 in 115.5 A generates a relative twist of 15 degrees between neighbouring b-strands if there are 24 b-strands in the helical repeat.

Most b-sheet structures in folded proteins are twisted rather than planar and have a right-handed twist of 0 to 30 degrees between strands. The right-handed twisted conformation represents the lowest energy conformation of the b-sheet structure (Pauling & Corey, 1951; Chothia, 1973). This model of the amyloid protofilament core incorporates the most likely, lower energy, right-handed twisted b-sheets but the data do not differentiate between left and right-handed helices. In this model, the twisting of the b-sheets around a common helical axis, which is parallel to the axis of the protofilament, accounts for the repeating unit of approximately 115.5 A that is observed in all the amyloid fibrils studied here.

The model is therefore an elaboration of the classical cross-b molecular structure, which permits the incorporation of the favourable twisted b-sheet structures (Pauling & Corey, 1951; Chothia, 1973). The helical structure of the protofilament enables the hydrogen bonding between the b-strands to be extended over the total length of the amyloid fibrils, thereby accounting for their characteristic rigidity and stability. The extended order in this dimension of the fibrils is responsible for the pseudo-crystalline sharpness of the meridional reflections.

The present X-ray results support the view that this model represents the core molecular structure of all of the amyloid fibrils studied here, irrespective of the number or arrangement of protofilaments, and demonstrate the independence of the 115.5 A helical repeat from the nature of precursor protein.

The ability of this single structure to accommodate different length polypeptide chains may be understood in the following way. Very short peptide chains, say six to ten residues, are able to form a single b-strand, which can act as the basic unit to be repeated along the fibre axis. Longer polypeptides will be able to form a larger number of b-strands by folding their chains back and forth. In this way the cross-b amyloid structure may be independent of the length of the polypeptide chains forming it.

[Note that protein subunit periodicity is then determined by the 24 strands per rotation: if there are 6 strands per subunit, then 4 subunits per rotation; the number of strands per subunit need not divide 24. Also, the subunits themselves may form an in-line face-to-face dimer that is the asymmetric unit. The model here does not distinuish parallel from anti-parallel beta structure; congo red favors anti-parallel inter-subunit joints. Chaperone involvement would favor the lower energy right-handed twist. -- webmaster]

The features of the structure that may vary and be dependent on characteristics of the precursor are mainly expressed in directions perpendicular to the fibre axis, where loops of varying length or other structures can be accommodated without affecting the core b-sheet structure. These variations would be expected to be reflected in variability of the spacings and intensities of the equatorial reflections. In contrast, the common b-sheet helical structure should result in a constant pattern for the spacings and intensities of meridional reflections. These characteristics are exactly what is observed in the diffraction patterns from different amyloid fibrils.

Fibrillogenesis and a structural conversion

Table 1 lists the known or predicted structures of the amyloid fibril subunit precursors in their non-fibrillar form. The amyloidogenic proteins display a wide range of native folds, yet the present analysis has demonstrated that all amyloid fibrils have the same cross-b molecular skeleton. Proteins such as the immunoglobulin light chain (Schormann et al., 1995), transthyretin (Blake et al., 1978; Terry et al., 1993; Hamilton et al., 1993; Sebastia o et al., 1996) and b2 -microglobulin (Becker & Reeke, 1985) have similar, mainly b-sheet native structures but, even so, must sustain significant structural changes when they are deposited in the cross-b amyloid form (Blake & Serpell, 1996; Blake et al., 1996), and it is known that the form of transthyretin that is amyloidogenic has a non-native conformation (Colon & Kelly, 1992; McCutchen et al., 1993, 1995; Kelly, 1996).

Proteins such as insulin (Adams et al., 1969), cystatin C (Bode et al., 1988), the amyloidogenic variants of lysozyme (Pepys et al., 1993; Booth et al., 1997), and the prion protein (Riek et al., 1996), which have extensive native a-helical structure, may undergo even larger conformational changes when they form amyloid fibrils.

Such a structural conversion has been demonstrated for the amyloidogenic variants of human lysozyme (Booth et al., 1997), which show an increase in b-sheet content and a loss of a-helical structure during fibril formation in vitro, and it is also associated with infectivity in the prion spongiform encephalopathies (Pan et al., 1993; Gasset et al., 1992, 1993; Harrison et al., 1997). Studies of various peptides corresponding to regions of the Alzheimer's disease Ab peptide have also demonstrated that structural plasticity is related to fibril formation (Hilbich et al., 1991; Barrow et al., 1992; Talafous et al., 1994; Sticht et al., 1995; Soto et al., 1995).

The present work demonstrates that, although the amyloidogenic proteins have very different precursor structures, they can all undergo a structural conversion, perhaps along a similar pathway, to a misfolded form that is the building block of the b-sheet helix protofilament. This mechanism of structural conversion and the generic structure of the amyloid protofilament offer two distinct targets for therapeutic molecules: compounds that could interfere with the transition from precursor to b-structured fold and agents that might inhibit or reverse the packing of protofilaments into fibrils. [Except that protofilaments themselves might be toxic. How either fibrils or protofiliments cause damage, if indeed they do, is currently the largest gap in understadnding -- webmaster]


We thank Drs G. A. Tennent, V. Bellotti and W. L. Hutchinson for preparing fibrils, and S. Lee for assistance with preparation of Figures. We thank Professor E. Lundgren and Dr O. Sangren, University of Umea , Sweden, for providing variant transthyretin Val30Met fibrils extracted from vitreous humor. L.C.S. was supported by the Oxford Centre for Molecular Sciences, and P. E. F. by the Alzheimer's Society of Ontario and the Ontario Mental Health Foundation. This work was supported in part by MRC Programme grant (G7900510) to M.B.P. and MRC Project grants to M.B.P. and C.C.F.B. We dedicate this paper to the memory of the late Dr George Glenner, the champion of b-fibrillosis.


Adams, M. J., Blundell, T. L., Dodson, E. J., Dodson, G. G., Vijayan, M., Baker, E. N., Harding, M. M., Hodgkin, D. C., Rimmer, B. & Sheat, S. (1969). The structure of rhombohedral 2 zinc insulin crystals. Nature, 224, 491495.

Arnott, S., Dover, S. & Elliot, A. (1967). Structure of b-poly-L-alanine: refined atomic co-ordinates for an anti-parallel beta-pleated sheet. J. Mol. Biol. 30, 201 208.

Barrow, C. J., Yasuda, A., Kenny, P. T. M. & Zagorski, M. G. (1992). Solution conformations and aggregational properties of synthetic amyloid b-peptides of Alzheimer's disease. J. Mol. Biol. 225, 10751093.

Becker, J. & Reeke, G. (1985). Three-dimensional structures b2 -microglobulin. Proc. Natl Acad. Sci USA, 82, 42254229.

Blake, C. C. F. & Serpell, L. C. (1996). Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous b-sheet helix. Structure, 4, 989998.

Blake, C. C. F., Geisow, M. J., Oatley, S. J., Rerat, B. & Rerat, C. (1978). Structure of prealbumin: secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 A . J. Mol. Biol. 121, 339356.

Blake, C. C. F., Serpell, L. C., Sunde, M. & Lundgren, E. (1996). A molecular model of the amyloid fibril. In CIBA Symposium No. 199, The Nature and origin of Amyloid Fibrils, pp. 621, John Wiley & Sons Ltd, Chichester, UK.

Bode, W., Engh, R., Musil, D., Thiele, U., Huber, R., Karshikov, A., Brzin, J., Kos, J. & Turk, V. (1988). The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J. 7, 25932599.

Bonar, L., Cohen, A. S. & Skinner, M. (1967). Characterization of the amyloid fibril as a cross-b Protein. Proc. Soc. Expt. Biol. Med. 131, 13731375.

Booth, D. R., Soutar, A. K., Hawkins, P. N., Reilly, M., Harding, A. & Pepys, M. B. (1994). Three new amyloidogenic transthyretin gene mutations: advantages of direct sequencing. In Amyloid and Amyloidosis 1993 (Kisilevsky, R., Benson, M. D., Frangione, B., Gauldie, J. T., Muckle, J. & Youngs, I. D., eds), pp. 456458, Parthenon Publishing, New York, Pearl River.

Booth, D. R., Sunde, M., Bellotti, V., Robinson, C. V., Hutchinson, W. L., Fraser, P. E. et al. (1997). Instability, unfolding and fibrillogenesis in amyloidogenic lysozyme variants. Nature, 385, 787 793.

Bradbury, E. M., Brown, L., Downie, A. R., Elliott, A., Fraser, R. D. B., Hanby, W. E. & Macdonald, T. R. R. (1960). The ``cross-beta'' structure in polypeptides of low molecular weight. J. Mol. Biol. 2, 276.

Burge, R. E. (1959). X-ray scattering by bundles of cylinders. Acta Crystallog. 12, 285289.

Burge, R. E. (1963). Equatorial X-ray diffraction by fibrous proteins: short range order in collagen, feather keratin and f-actin. J. Mol. Biol. 7, 213224. Burke, M. J. & Rougvie, M. A. (1972). Cross-b protein structures I. Insulin fibrils. Biochemistry, 11, 2435 2439.

Burtnick, L. D., Robinson, R. C. & Koepf, E. K. (1996). The structure of horse plasma gelsolin to 2.5 A . Biophys. J. 70, Pt. 2, pSUA12.

Chothia, C. (1973). Conformations of twisted b-sheets in proteins. J. Mol. Biol. 75, 295302.

Cohen, A. S., Shirahama, T. & Skinner, M. (1981). Electron microscopy of amyloid. In Electron Microscopy of Protein (Harriss, I., ed.), vol. 3, pp. 165205, Academic Press, London.

Colon, W. & Kelly, J. W. (1992). Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry, 31, 86548660.

Come, J. H., Fraser, P. E. & Lansbury, P. T. (1993). A kinetic model for amyloid formation in the prion diseases: importance of seeding. Proc. Natl Acad. Sci. USA, 90, 59595963.

Damas, A., Sebastiao, M. P., Domingues, F. S., Costa, P. P. & Saraiva, M. J. (1995). Structural studies on FAP fibrils: removal of contaminants is essential for the interpretation of X-ray data. Amyloid: Int. J. Exp. Clin. Invest. 2, 1731278.

Eanes, E. D. & Glenner, G. G. (1968). X-ray diffraction studies on amyloid filaments. J. Histochem. Cytochem, 16, 673677.

Filshie, B. K., Fraser, R. D. B., MacRae, T. P. & Rogers, G. E. (1964). X-ray diffraction and electron microscope observations on soluble derivatives of keratin. Biochem. J. 92, 1926.

Fraser, P. E., Nguyen, J. T., Surewicz, W. K. & Kirschner, D. A. (1991). pH dependent structural transitions of Alzheimer's amyloid peptides. Biophys. J. 60, 11901201.

Gasset, M., Baldwin, M. A., Lloyd, D. H., Gabriel, J.-M., Holtzman, D. M., Cohen, F. E., Fletterick, R. & Prusiner, S. B. (1992). Predicted a-helical regions of the prion protein, when synthesized as peptides, form amyloid. Proc. Natl Acad. Sci. USA, 89, 10940 10944.

Gasset, M., Baldwin, M., Fletterick, R. & Prusiner, S. (1993). Perturbation of secondary structure of the scrapie prion protein under conditions that alter infectivity. Proc. Natl Acad. Sci. USA, 90, 15.

Geddes, A. J., Parker, K. D., Atkins, E. D. T. & Beighton, E. (1968). ``Cross b`` conformation in protein. J. Mol. Biol. 32, 343358.

Gilchrist, P. & Bradshaw, J. (1993). Amyloid formation by salmon calcitonin. Biochim. Biophys. Acta, , 111 114.

Glenner, G. G. (1080a). Amyloid deposits and amyloidosis. The beta-fibrilloses (part one). New Eng. J. Med. 302, 12831292.

Glenner, G. G. (1980b). Amyloid deposits and amyloidosis. The bea-fibrilloses (part two). New Eng. J. Med. 302, 13331343.

Glenner, G. G. & Wong, C. W. (1984). Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 120, 885890.

Gorevic, P., Castano, E., Sarma, R. & Frangione, B. (1987). Ten to fourteen residue peptides of Alzheimer's disease protein are sufficient for amyloid fibril formation and its characteristic X-ray diffraction pattern. Biochem. Biophys. Res. Commun. 147, 854862.

Hamilton, J., Steinrauf, L., Braden, B., Liepnieks, J., Benson, M., Holmgren, G., Sandgren, O. & Steen, L. (1993). The X-ray crystal structure refinements of normal human transthyretin and the amyloidogenic Val-30-Met variant to 1.7 A resolution. J. Biol. Chem. 268, 24162424.

Harrison, P. M., Bamborough, P., Daggett, V., Prusiner, S. B. & Cohen, F. E. (1997). The prion folding problem. Curr. Opin. Struct. Biol. 7, 5359.

Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. & Beyreuther, K. (1991). Aggregation and secondary structure of synthetic amyloid bA4 peptides of Alzheimer's disease. J. Mol. Biol. 218, 149163.

Inouye, H., Fraser, P. E. & Kirschner, D. A. (1993). Structure of b-crystallite assemblies by Alzheimer b-amyloid protein analogues: analysis by X-ray diffraction. Biopys. J. 64, 502519.

Jarvis, J. A., Craik, D. J. & Wilce, M. C. J. (1993). X-ray diffraction studies of fibrils formed from peptide fragments of transthyretin. Biochem. Biophys. Res. Commun. 192, 991998.

Kelly, J. W. (1996). Alternative conformations of amyloidogenic proteins govern their behaviour. Curr. Opin. Struct. Biol. 6, 1117.

Kelly, J. W. (1997). Amyloid fibril formation and protein misassembly: a structural quest for insights into amyloid and prion diseases. Structure, 5, 595 600.

Kirschner, D. A., Abraham, C. & Selkoe, D. A. (1986). X-ray diffraction from intraneuronal paired helical filaments and extra-neuronal amyloid fibres in Alzheimer's disease indicates crossb conformation. Proc. Natl Acad. Sci. USA, 83, 503507.

Kirschner, D. A., Inouye, H., Duffy, L., Sinclair, A., Lind, M. & Selkoe, D. A. (1987). Synthetic peptide homologous to b-protein from Alzheimer's disease forms amyloid-like fibrils in vitro. Proc. Natl Acad. Sci. USA, 84, 69536957.

Lorenz, M. & Holmes, K. C. (1993). Computer processing and analysis of X-ray diffraction data. J. Appl. Crystallog. 26, 8291.

McCutchen, S., Colon, W. & Kelly, J. W. (1993). Transthyretin mutation Leu-55-Pro significantly alters tetramer stability and increases amyloidogenicity. Biochemistry, 32, 1211912127.

McCutchen, S. L., Lai, Z., Miroy, G. J., Kelly, J. W. & Colon, W. (1995). Comparison of lethal and nonlethal transthyretin variants and their relationship to amyloid disease. Biochemistry, 34, 1352713536.

Nelson, S., Lyon, M., Gallager, J., Johnson, E. & Pepys, M. B. (1991). Isolation and characterisation of the integral glycosaminoglycan constitutents of human amyloid A and monoclonal light-chain amyloid fibrils. Biochem. J. 275, 6774.

Nguyen, J. T., Inouye, H., Baldwin, M. A., Fletterick, R., Cohen, F. E., Prusiner, S. B. & Kirschner, D. A. (1995). X-ray diffraction from scrapie prion rod and PrP peptides. J. Mol. Biol. 252, 412422.

Nolte, R. T. & Atkinson, D. (1992). Conformational analysis of apolipoprotein A-1 and E-3 based on primary sequence and circular dichroism. Biophys. J. 63, 12211239.

Pan, K.-M., Baldwin, M. A., Nguyen, J. T., Gaseet, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E. & Prusiner, S. B. (1993). Conversion of a-helices into b-sheets features in the formation of the scrapie prion proteins. Proc. Natl Acad. Sci. USA, 90, 1096210966.

Pauling, L. & Corey, R. (1951). Configuration of polypeptide chains with favoured orientation around single bonds: two new pleated sheets. Proc. Natl Acad. Sci. USA, 37, 729739.

Pepys, M. B. (1996). Amyloidosi. In The Oxford Textbook of Medicine (Weatherall, D. J., Ledingham, J. G. G. & Warell, D. A., eds), 3rd edit., vol. 2, pp. 15121524, Oxford University Press, Oxford.

Pepys, M. B., Hawkins, P. N., Booth, D. R., Vigushin, D. M., Tennet, G. A., Soutar, A. K., Totty, N., Nguyen, O., Blake, C. C. F., Terry, C. J., Feest, T. G., Zalin, A. M. & Hsuan, J. J. (1993). Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature, 362, 553557.

Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R. & Wu thrich, K. (1996). NMR structure of the mouse prion protein domain PrP(121 231). Nature, 382, 180182.

Schormann, N., Murrell, J. R., Liepnieks, J. & Benson, M. (1995). Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation. Proc. Natl Acad. Sci. USA, 92, 94909494.

Sebastiao, P., Dauter, Z., Saraiva, M. J. & Damas, A. M. (1996). Crystallization and preliminary X-ray diffraction studies of Leu55Pro variant transthyretin. Acta Crystallog. sect. D, 52, 566568.

Serpell, L. C., Sunde, M., Fraser, P. E., Luther, P. K., Morris, E., Sandgren, O., Lundgren, E. & Blake, C. C. F. (1995). The examination of the structure of the transthyretin amyloid fibril by image reconstruction from electron micrographs. J. Mol. Biol. 254, 113118.

Shirahama, T. & Cohen, A. S. (1967). High resolution electron microscopic analysis of the amyloid fibril. J. Cell Biol, 33, 679706.

Shirahama, T., Benson, M. D., Cohen, A. S. & Tanaka, A. (1973). Fibrillar assemblage of variable segments of immunoglobulin light chains: an electron microscopic study. J. Immunol. 110, 2130.

Soto, C., Castano, E., Frangione, B. & Inestrosa, N. (1995). The a-helical to b-sheet transition in the amino-terminal fragment of the amyloid b-peptide modulates amyloid formation. J. Biol. Chem. 270, 30633067.

Sticht, H. P., Bayer, P., Willbold, D., Dames, S., Hilbich, C., Beyreuther, K., Frank, R. & Rosch, P. (1995). Structure of amyloid A4(1-40)-peptide of Alzheimer's disease. Eur. J. Biochem. 233, 293298.

Soutar, A. K., Hawkins, P. N., Vigushin, D. M., Tennent, G. A., Booth, S., Hutton, T., Nguyen, O., Totty, N., Feest, T. G., Hsuan, J. J. & Pepys, M. B. (1992). Apolipoprotein A-1 mutation Arg-60 causes autosomal dominant amyloidosis. Proc. Natl Acad. Sci. USA, 89, 73897393.

Tagliavini, F., Prelli, F., Verga, L., Giaccone, G., Jarma, R., Gorevic, P., Ghetti, B., Passerini, F., Ghibaudi, E., Forloni, G., Salmona, M., Bugiani, O. & Frangione, B. (1993). Synthetic peptides homologous to prion protein residues 106147 form amyloidlike fibrils in vitro. Proc. Natl Acad. Sci. USA, 90, 96789682.

Talafous, J., Marcinowski, K., Klopman, G. & Zagorski, M. (1994). Solution structure of residues 128 of the amyloid b-peptide. Biochemistry, 33, 77887796. Tan, S. Y., Pepys, M. B. & Hawkins, P. N. (1995). Treatment of amyloidosis. Am. J. Kidney Dis. 26, 267285.

Terry, C. J., Damas, A. M., Oliviera, P., Saraiva, M. J. M., Alves, A. L., Costa, P. P., Matias, P. M., Sakaki, Y. & Blake, C. C. F. (1993). Structure of Met30 variant of transthyretin and its amyloidogenic variations. EMBO J. 12, 735741.

Turnell, W., Sarra, R., Baum, J. O., Caspi, D., Baltz, M. L. & Pepys, M. B. (1986a). X-Ray scattering and diffraction by wet gels of AA amyloid fibrils. Mol. Biol. Med. 3, 409424.

Turnell, W., Sarra, R., Glover, I. D., Baum, J. O., Caspi, D., Baltz, M. L. & Pepys, M. B. (1986b). Secondary structure preduction of human SAA1 , presumptive identification of calcium and lipid binding sites. Mol. Biol. Med. 3, 387407.

Worcester, D. L. (1978). Structural origins of diamagnetic anisotropy in proteins. Proc. Natl Acad. Sci. USA, 75, 54755477.

Edited by F. E. Cohen
(Received 8 May 1997; received in revised form 5 August 1997; accepted 5 August 1997)

A Conformational Transition at the N Terminus of the Prion Protein Features in Formation of the Scrapie Isoform.

J Mol Biol 1997 Oct 31;273(3):614-622 fulltext free as pdf
Peretz D, Williamson RA, Matsunaga Y, Serban H, Pinilla C, Bastidas RB, 
Rozenshteyn R, James TL, Houghten RA, Cohen FE, Prusiner SB, Burton DR
The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27-30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27-30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27-30. Denaturation of PrP 27-30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27-30.

A larger view of this image is available at the Prion Image Gallery. The image summarizes epitope locations from Oesch et al, this paper, and a recent PNAS paper on 'protein x' interacting residues.

The data reported here provide the frst glimpse of the localized conformational changes that PrP C undergoes as it is converted into PrP Sc . One approach to probing conformational rearrangements in prion proteins is to raise antibodies to diverse epitopes of PrP C and PrP Sc .A panel of monoclonal Fab fragments was obtained that recognized PrP Sc denatured with 3 M guanidinium thiocyanate (GdnSCN) both in vitro and in situ, as well as recombinant PrP spanning residues 90 to 231. All but one of the antibodies also bound to PrP C on the cell surface as determined by flow cytometry. Disappointingly, however, PrP 27-30 was not detected by any of these antibodies prior to treatment with GdnSCN.

An explanation for this fnding may lie in the physicochemical properties and antigenicity of PrP 2730 in prion rods. During purifcation of PrP Sc , brain homogenate was subjected to digestion with proteinase K and extraction with 0.5% Sarkosyl. Purifed PrP 27-30 polymerizes into rod-like structures of 100 to 200 nm in length that are composed of as many as 1000 PrP 27-30 molecules (McKinley et al., 1991; Prusiner et al., 1983). This aggregation may reduce effective epitope concentrations, thereby hindering effcient immunization and selection of specifc antibody phage. We have previously shown that epitope concentration is a critical factor in effective selection from antibody phage display libraries (Parren et al., 1996).

In the study reported here, we have attempted to overcome the possible problem of cryptic epitopes in PrP 27-30 within prion rods by immunizing mice with a dispersed form of PrP 27-30 (Gabizon et al., 1987) and rescuing the corresponding recombinant antibodies. The dispersed form of PrP 27-30 is prepared by sonication, solubilization in detergent, and incorporation into liposomes (Gabizon et al., 1987). The procedure does not result in any loss of scrapie infectivity. Prnp 0/0 mice immunized with dispersed PrP 27-30 were used to generate a new panel of recombinant antibodies, some of which react with nondenatured PrP 27-30. A comparison of the reactivity of antibodies with PrP C with that with nondenatured PrP 27-30 reveals that conformational differences between the two molecules lie within the N-terminal region corresponding to residues 90 to 120.


Our earlier studies had described Fabs that recognize denatured SHaPrP 27-30 and native PrP C , but not nondenatured PrP 27-30 (Williamson et al., 1996).

We now report on the ability of new recombinant Fabs to detect non-denatured PrP 27-30 by ELISA and by immunoprecipitation assays. Except for D13 and D4, the Fabs reacted equally well with rPrP in ELISA, whether or not the antigen was treated with GdnSCN (Figure 1(a)). All of the recombinant Fabs, together with the hybridoma-derived 13A5 and 3F4 mAbs (Barry & Prusiner, 1986; Kascsak et al., 1987), also reacted strongly with PrP 27-30 rods that had been coated onto ELISA wells and then exposed to 3 M GdnSCN (Figure 1(b)). We determined whether each of the antibodies was able to immunoprecipitate PrP C from transfected CHO cells expressing SHaPrP C.

...When taken together these data support the argument that following exposure to denaturant, the conformation of PrP 27-30 is disturbed and upon removal of the denaturant, the protein adopts a PrP C -like conformation.

Cryptic and exposed epitopes on PrP 27-30

We compared the immunoreactivity of recombinant Fabs with native and GdnSCN-treated PrP 27-30 by ELISA (Figure 3). Although our data suggest that GdnSCN-treated PrP 27-30 refolds to a PrP C -like molecule, we refer to this GdnSCNtreated protein as denatured for convenience and clarity. To expose hidden epitopes, PrP 27-30 rods were dispersed into liposomes, biotinylated, and captured onto the surface of streptavidin-coated ELISA wells. Three Fabs designated R10, D13, and D4 bound weakly to native PrP 27-30 but reacted strongly with denatured PrP 27-30. In contrast, the three Fabs designated R1, R2, and D2 bound almost as well to native PrP 27-30 as to denatured PrP 27-30. The Fab designated R72 gave a pattern of immunoreactivity that was intermediate between the two groups of Fabs described above. The R10, D13, and D4 Fabs showed a pattern of immunoreactivity with native and denatured PrP 27-30 similar to that found for the 3F4 Fab, which binds to PrP residues 109 to 112 (Rogers et al., 1991). The R72 Fab demonstrated a pattern of immunoreactivity with native and denatured PrP 27-30 similar to that found for the 13A5 Fab, which binds to PrP residues 138 to 141 (Rogers et al., 1991).

To extend the results of our ELISA studies, we examined two Fabs with respect to immunoprecipitation of PrP 27-30 incorporated into liposomes. Previously, no PrP-specifc antibodies, either those derived from hybridoma fusions or those from phage libraries, were able to immunoprecipitate PrP 27-30, unless it was frst treated with GdnSCN (Borchelt et al., 1992; Taraboulos et al., 1990, 1992). In striking contrast to those earlier studies, 50 ng of the R2 Fab effciently precipitated native PrP 27-30 (Figure 4). As in the earlier studies noted above, neither the D4 nor the 3F4 Fab was able to immunoprecipitate PrP 27-30. Even 500 ng of the D4 or 3F4 Fab was insuffcient to immunoprecipitate native PrP 27-30 dispersed into liposomes as measured by Western blotting. Identification of epitopes

... The excellent correlation between the epitopes of the Fabs and the previously known epitopes of the hybridoma-derived IgG 3F4 and 13A5 mAbs (Rogers et al., 1991) with respect to immunoreactivity with native and denatured PrP 27-30 supports our conclusion that an N-terminal conformational transition features in the formation of PrP Sc .


Our results with PrP-specifc Fabs provide a structural map of the conformational transition that features in the formation of PrP Sc . These fndings, in concert with information from other recent studies, demonstrate that the N-terminal region of PrP 27-30 from residue 90 to 120 is critical for PrP Sc formation (Muramoto et al., 1996). Deletion of any portion of this region prevents the acquisition of protease resistance, a property that is often but not always characteristic of PrP Sc . Synthetic peptides corresponding to this region of PrP exhibit considerable conformational flexibility consistent with the a-helix to b-sheet transition (Zhang et al., 1995) that PrP C undergoes when it is transformed into PrP S.

The conformational plasticity of this region is further emphasized by the recent fndings that two distinct prion strains exhibit different sites of proteolytic cleavage within this region (Bessen & Marsh, 1994; Telling et al., 1996). These fndings are in accord with molecular modeling studies that predicted that the N-terminal region of PrP 27-30 was likely to be the site where a-helical structures were transformed into b-sheets as PrP Sc was formed (Huang et al., 1994, 1996).

...The studied C terminus epitope region (225 to 231) is adjacent to SHa residues 215 and 219, which are at the binding site of a presumed protein, designated protein X, that mediates PrP Sc formation (Kaneko et al., 1997). Our fnding that the C termini of PrP C and PrP Sc are conformationally similar as judged by immunoreactivity with Fabs is of particular interest with respect to protein X, since this protein seems to bind PrP C but not PrP Sc (Telling et al., 1995).

In summary, we report for the frst time an epitope region at the C terminus that is exposed in both PrP C and PrP Sc. In contrast, epitopes towards the N terminus of the protein that are largely cryptic in PrP Sc are exposed in PrP C.

Prion Protein Aggregation Reverted by Low Temperature in Transfected Cells Carrying a Prion Protein Gene Mutation.

J Biol Chem 1997 Nov 7;272(45):28461-28470 
Singh N, Zanusso G, Chen SG, Fujioka H, Richardson S, Gambetti P, Petersen RB
Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC), a glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into an isoform that is protease-resistant (PrPres) and pathogenic. In inherited prion diseases, mutations in the prion protein (PrPM) engender the conversion of PrPM into PrPres. We developed a cell model of GSS disease, a neurodegenerative condition characterized by PrPM-containing amyloid deposits and neuronal loss, by expressing the GSS haplotype Q217R-129V in human neuroblastoma cells. By comparison to PrPC, this genotype results in the following alterations of PrPM:

1) expression of an aberrant form lacking the glycosylphosphatidylinositol anchor,
2) increased aggregation and protease resistance, and
3) impaired transport to the cell surface. Most of these alterations are temperature-sensitive, indicating that they are due to misfolding of PrPM.

The affected gene for GPI surface protein defect codes for the GPI transamidase

 Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 12580-12585, November 1997
Jianliang Yu, S. Nagarajan,, J J. Knez*, Sidney Udenfriend, Rui Chen*, and M. Edward Medof
The final step in GPI anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins.

In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. Studies with yeast have shown that at least two gene products are involved in the final C-terminal GPI transamidation step.

The first of these genes to be identified, termed GAA1 (20), was isolated from a Saccharomyces cervisiae mutant (gaa1) that initially was noted to be defective in endocytosis. It subsequently was found to synthesize the fully assembled yeast GPI precursor CP2 but, unlike wild-type cells, failed to transfer it to the proform of GPI-anchored gas1p (21). It is a 614-amino acid-long multimembrane-spanning ER protein with a large lumenal domain near its cytoplasmically oriented N terminus.

A human homolog of the protein has been identified that has 28% overall but greater amino acid homology in the lumenal domain. The second of these proteins, yGPI8, the human homolog that is the subject of this report, was recently cloned from a mutagenized S. cervisiae clone gpi-8 that exhibited a similar phenotype (22). The yGPI8 gene encodes a 47-kDa 411-amino acid-long type I transmembrane ER protein which has a large N-terminal luminal domain and a short cytosolic C-terminal domain of 14 amino acids. The functions of yeast gaa1p and of gpi8p are unknown. The observation (23) that yeast gpi8p has 27.5% identity to a jackbean asparaginyl endopeptidase, which has been found to exhibit transamidase activity in vitro, has suggested that this protein is the transamidase itself. Previously characterized transamidases do not have cofactors nor are they composed of more than one subunit (19, 24). Our finding that hGPI8 restores the ability of class K microsomes to support nascent protein uptake of GPI and interaction with the potent nucleophile HDZ concomitant with cleavage of the protein at its site provides fairly convincing evidence that hGPI8 codes for the GPI transamidase. The development of a reconstituted system with purified proteins (transamidase complex + proprotein + GPI) would establish this firmly.

pH-dependent Stability and Conformation of the Recombinant Human Prion Protein PrP(90-231).

Swietnicki W, Petersen R, Gambetti P, Surewicz WK
J Biol Chem 1997 Oct 31;272(44):27517-27520 
A recombinant protein corresponding to the human prion protein domain encompassing residues 90-231 (huPrP(90-231)) was expressed in Escherichia coli in a soluble form and purified to homogeneity. Spectroscopic data indicate that the conformational properties and the folding pathway of huPrP(90-231) are strongly pH-dependent. Acidic pH induces a dramatic increase in the exposure of hydrophobic patches on the surface of the protein. At pH between 7 and 5, the unfolding of hPrP(90-231) in guanidine hydrochloride occurs as a two-state transition. This contrasts with the unfolding curves at lower pH values, which indicate a three-state transition, with the presence of a stable protein folding intermediate. While the secondary structure of the native huPrP(90-231) is largely alpha-helical, the stable intermediate is rich in beta-sheet structure. These findings have important implications for understanding the initial events on the pathway toward the conversion of the normal into the pathological forms of prion protein.

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