Conformational Transformations in Peptides Containing Two Putative α-Helices of the Prion Protein

JMB—MS 634 Cust. Ref. No. CAM 105/95

[SGML] J. Mol. Biol. (1995) 250, 514–526

Conformational Transitions in Peptides Containing Two Putative a-Helices of the Prion Protein Hong Zhang1, Kiyotoshi Kaneko2, Jack T. Nguyen2, Tatiana L. Livshits2 Michael A. Baldwin2, Fred E. Cohen1,3,4, Thomas L. James1 and Stanley B. Prusiner2,4* Departments of Pharmaceutical Chemistry 2 Neurology, 3Medicine and 4 Biochemistry & Biophysics University of California San Francisco, CA 94143-0518, USA

Prions are composed largely, if not entirely, of the scrapie isoform of the prion protein (PrPSc). Conversion of the cellular isoform (PrPC) to PrPSc is accompanied by a diminution in the a-helical content and an increase in the b-sheet structure. To investigate the structural basis of this transition, peptide fragments corresponding to Syrian hamster PrP residues 90 to 145 and 109 to 141, which contain the most conserved residues of the prion protein and the first two putative a-helical regions in a PrPC model, were studied using infrared spectroscopy and circular dichroism. The peptides could be induced to form a-helical structures in aqueous solutions in the presence of organic solvents, such as trifluoroethanol and hexafluoroisopropanol, or detergents, such as sodium dodecyl sulfate and dodecyl phosphocholine. NaCl at physiological concentration or acetonitrile induced the peptides to acquire substantial b-sheet. The intermolecular nature of the b-sheet was evident in the formation of rod-shaped polymers as detected by electron microscopy. Resistance to hydrolysis by proteinase K and epitope mapping argue that the b-sheet structures were formed by the interaction of residues lying between 109 and 141. A similar range of residues was shown by nuclear magnetic resonance spectroscopy to be capable of forming a-helices. The a-helical structures seem to require a hydrophobic support from either intermolecular interactions or the hydrophobic environment provided by micelles, in agreement with the predicted hydrophobic nature of the packing surface among the four putative helices of PrPC and the outer surfaces of the first two helices. Our results suggest that perturbation of the packing environment of the highly conserved residues is a possible mechanism for triggering the conversion of PrPC to PrPSc where a-helices appear to be converted into b-sheets.

*Corresponding author

Keywords: prion protein; conformational transition; a-helix; b-sheet; NMR

1

Introduction Prion diseases seem to be the first recognized disorders of protein conformation (Cohen et al., 1994; Prusiner, 1994a). Prions cause fatal neurodegenerative illness of animals and humans. More than 130,000 cattle have died of ‘‘mad cow’’ disease in Great Britain where prions are thought to have contaminated meat and bone meal fed to the cattle as a dietary supplement (Wilesmith, 1994). A change in the method used to render sheep offal appears to have led to the production of prion-contaminated feed since scrapie, another prion disease, was endemic in sheep in Britain. In humans, a quartet of neurologic disorders caused by prions have been identified: kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Stra¨ussler–Scheinker disease (GSS), and 0022–2836/95/290514–13 $08.00/0

fatal familial insomnia (FFI) (Gajdusek, 1977; Prusiner, 1994a). Remarkably, familial CJD, GSS and FFI are inherited illnesses that are caused by mutations in the prion protein (PrP) gene yet they are also transmissible to experimental animals (Tateishi et al., 1992; Brown et al., 1994). The prion protein (PrP) was discovered by enriching fractions from Syrian hamster brains for scrapie prion infectivity (Prusiner et al., 1982). Although prions are composed largely, if not entirely, of the diseased isoform, PrPSc, they readily transmit disease to inoculated recipients (Prusiner, 1991). PrPSc differs from its cellular isoform, PrPC, in that (1) PrPSc accumulates in the cell while PrPC turns over rapidly from the cell surface (Borchelt et al., 1990); (2) PrPSc is partially resistant to proteinase digestion, forming a 27 to 30 kDa fragment designated PrP 7 1995 Academic Press Limited

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Figure 1. A graphic representation of peptides 90-145, 109-141, 109-122, 129-141 and 104-122 of the Syrian hamster prion protein (SHa PrP). The residues corresponding to the first two putative helices H1 and H2 (Gasset et al., 1992; Huang et al., 1994) are underlined.

27-30, whereas PrPC is completely degraded (Oesch et al., 1985); and (3) PrPSc is insoluble, while PrPC can be dissolved in detergents (Meyer et al., 1986). While attempts to identify a post-translational chemical process responsible for the conversion of PrPC into PrPSc were unsuccessful (Stahl et al., 1993), spectroscopic studies showed that PrPC contains 040% a-helical structure with little b-sheet (Pan et al., 1993). In contrast, PrPSc and its protease resistant core are rich in b-sheet (Prusiner et al., 1983; Caughey et al., 1991; Gasset et al., 1993; Pan et al., 1993; Safar et al., 1993). Molecular modeling studies suggested that PrPC might fold into a four a-helix bundle protein and that one or more of the putative a-helices might refold into b-sheets upon conversion of PrPC into PrPSc (Gasset et al., 1992; Huang et al., 1994). Ten of 11 known point mutations that segregate with the inherited human prion diseases lie either within or adjacent to these putative regions of secondary structure (Huang et al., 1994; Prusiner, 1994b). Amino acid substitutions in the PrP gene due to species variation generally occur outside these regions of presumed secondary structure (Scha¨tzl et al., 1995). Synthetic peptides corresponding to three of the four putative a-helices were found to form b-sheets and polymerize into fibrils with the tinctorial characteristics of amyloid when dispersed in water (Gasset et al., 1992). Because transgenetic studies have indicated that PrPC and PrPSc form a complex during prion replication (Prusiner et al., 1990), we sought to simulate the interaction between these two PrP isoforms by mixing the peptide corresponding to the first putative a-helix H1 in a b-sheet conformation with another peptide in an a-helical or random coil conformation. Mixing of H1 with the extended H1 composed of residues 104 to 122 or the second putative a-helix H2 in aqueous buffers converted the conformations of these peptides into b-sheets (Nguyen et al., 1995). To study the H1 and H2 regions within the context of the PrP sequence, we constructed Syrian hamster (SHa) polypeptides in which both regions of putative secondary structure are contained extending from residue 90 to 145 and from 109 to 141. Peptide 90-145 was chosen because residue 90 is the N terminus of the infectious PrP 27-30 molecule (Prusiner et al., 1984) and residue 145 coincides with the stop codon in the PrP gene of a Japanese patient who died following a prolonged GSS illness (Kitamoto et al., 1993). Whether the PrP peptide 90-145 can be induced to adopt a conformation that will confer ‘‘infectivity’’ upon it remains to be determined.

A smaller peptide, 109-141, was also chosen because it extends from the N terminus of H1 to the C terminus of H2; in addition, shorter peptides 109-122 (H1), 104-122 and 129-141 (H2) previously investigated (Gasset et al., 1992) were also studied (Figure 1). It was found in this study that the peptides that contain both H1 and H2 could be induced to form a-helical structures in aqueous solutions in the presence of organic solvents, such as trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), or detergents, such as sodium dodecyl sulfate (SDS) and dodecyl phosphocholine (DPC). Sodium chloride at physiological concentration or acetonitrile (ACN) induced the same peptides to acquire substantial amounts of b-sheet. The intermolecular nature of the b-sheet was evident in the formation of rod-shaped polymers. Resistance to hydrolysis by proteinase K indicates that the b-sheet structures were formed by the interaction of residues lying between residues 109 and 141. A similar range of residues was shown by nuclear magnetic resonance (NMR) spectroscopy to be capable of forming a-helices. The a-helical structures seem to require a hydrophobic support from either intermolecular interactions or a hydrophobic environment provided by detergent micelles, in agreement with the predicted hydrophobic nature of the packing surface among the four putative helices of PrPC and the outer surfaces of the first two helices (Huang et al., 1994). The secondary structures of the PrP peptides as determined by NMR are consistent with the model of PrPC. Perturbation of the packing environment of the highly conserved residues is suggested as a possible mechanism for triggering the structural conversion of PrPC to PrPSc where a-helices appear to be converted into b-sheets. Our findings are consistent with the predicted PrPC structure, and they support the hypothesis of a conformational transition during the conversion of PrPC to PrPSc (Cohen et al., 1994).

Results Formation of a-helices The effect of pH on the formation of a-helical structures was found to be insignificant between pH 3.7 and 7.2 from examination of peptides 109-141 and 90-141 in aqueous solutions by CD and NMR. In an aqueous solution of 20 mM sodium acetate at pH 3.7, peptides 90-145, 109-141, 104-122 and 129-141 exhibited largely random coil structures with a characteristic negative CD band at 198 nm (data not

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Figure 2. The CD spectra of peptides 104-122, 129-141 and 90-145 in 20 mM sodium acetate at pH 3.7 with 10% (v/v) HFIP for peptide 90-145 and 50% (v/v) HFIP for peptides 104-122 and 129-141. Mean residue molar ellipticity is used as the vertical axis. (—), 90-145; (– – –), 104-122; (· · · ), 129-141.

shown). When HFIP or a detergent (SDS or DPC) was added to the same peptide solutions, a-helical structures were formed in some cases, with characteristic negative CD bands at 208 nm and 222 nm and a positive band at 192 nm (see Figures 2 and 3). Peptides 104-122, 129-141 and 90-145 required different concentrations of HFIP to form a-helical structures. As shown in Figure 2, only 10% HFIP was required to convert peptide 90-145 into an a-helical conformation, whereas at least 50% HFIP was needed to stabilize the a-helical structure of peptide 104-122. However, even 50% HFIP was found to be insufficient to force peptide 129-141 into an a-helix (Figure 2). Similarly, the peptides dispersed in the detergents displayed differential a-helix-forming propensities. In SDS, peptides 90-145, 109-141 and 104-122 were all stabilized into a-helices while peptide 129-141 was not (Figure 3(a)). In DPC, the long peptides 90-145 and 109-141, which contain both H1 and H2 (Gasset et al., 1992; Huang et al., 1994), formed a-helical structures while the shorter peptides did not (Figure 3(b)). These findings together with the findings in the HFIP solutions suggest that the putative H1 region (Gasset et al., 1992; Huang et al., 1994) is intrinsically better suited to an a-helical structure, and that the existence of the H1 helix may be important in promoting the folding of the H2 a-helical structure in the longer peptides. SDS is known to be more hydrophobic than DPC and was found to be more a-helix-promoting than DPC. This argues that the hydrophobic environment provided by the micelle interior is important in stabilizing the a-helical structures of the peptides.

Conformational Transitions of PrP Peptides

(a)

(b) Figure 3. The CD spectra of peptides 104-122, 129-141, 109-141 and 90-145 in (a) 100 mM SDS, 20 mM sodium acetate at pH 3.7 and (b) 100 mM DPC, 20 mM sodium acetate at pH 3.7. The vertical axis is mean residue molar ellipticity. (· · · ·), 104-122; (– · –), 129-141; (– – –), 109-141; (—), 90-145.

Aggregation of a-helical structures The ultrastructure of peptide 90-145 directly dissolved in an aqueous solution containing either SDS or HFIP, as shown in Figure 4, suggested that the peptide was not monomeric under these conditions. The peptide aggregates formed rods and ribbons of 20 to 30 nm in diameter and >500 nm in length in the HFIP solution. Examination of the peptide by NMR under the same solution conditions showed that the linewidths of the proton resonances were hetero-

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Figure 4. The ultrastructure of peptide 90-145 in 20 mM sodium acetate at pH 3.7 with A, 100 mM SDS and B, 10% HFIP. The bars represent 100 nm in length.

geneous and larger than expected for the peptide in monomeric form (data not shown). Because it is known that proton linewidths are proportional to the molecular mass of a molecule, this result confirmed intermolecular aggregation. Under the same conditions, peptides 109-141 (data not shown) and 109-122 (Nguyen et al., 1995) were found to form similar aggregates. By manipulating the solution conditions, we were able to disperse peptides 109-122, 109-141 and 90-145 into largely monomeric states in SDS micelles. This was achieved by initially dispersing the peptides and SDS in a 1:100 ratio into 50% (v/v) HFIP/H2 O to ensure the homogeneous mixing of the peptide and SDS molecules. The samples were then lyophilized and rehydrated. Under these conditions, the proton resonance linewidths in the NMR spectra were found to be smaller and more uniform than before (see Figure 5), indicating a smaller and more homogeneous molecular size for the peptides. These results suggest that a hydrophobic environment was required to maintain the monomeric a-helical structures. The intermolecular aggregation observed in both the HFIP and the detergents before the manipulation of solution conditions was therefore most likely driven by hydrophobic interactions. It is likely that, in addition to the contribution from the possible folding cooperativity between H1 and H2 in peptide 90-145, the hydrophobicity provided by the intermolecular aggregations may also contribute to the differential a-helix-forming propensities

displayed by peptides 104-122, 129-141 and 90-145 in the HFIP solutions. Secondary a-helical structures We have accomplished sequence-specific assignments of the NMR spectra for peptides 109-122, 109-141 and 90-145. The assignment of the shorter peptide 109-122 formed the basis for assigning the longer peptides. Figure 5 shows an NMR NOESY spectrum of the correlation between amide protons and a protons of peptide 109-141. The assignment and the chemical shift values of the peptide 109-141 are shown in Table 1. Previous studies have correlated protein secondary structures with chemical shift values and nuclear Overhauser enhancement (NOE) connectivities (Wu¨thrich, 1986; Wishart et al., 1992; Wishart & Sykes, 1994; Merutka et al., 1995; Wishart et al. 1995). Chemical shift values of a protons can be shifted upfield by 00.4 p.p.m in an a-helix and downfield by 00.4 p.p.m. in a b-sheet when compared with random coil values (criterion 1). Based on these changes, chemical shift indices of −1 (a-helix), 0 (random coil), and +1 (b-sheet) (Wishart et al., 1992; Wishart & Sykes, 1994) are defined to identify secondary structures (criterion 2). NOE connectivities can also be used in identifying secondary structures (criterion 3). An NOE cross-peak cor˚ and its responds to an interproton distance (r) E5 A intensity is proportional to 1/r 6. This correlation

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Figure 5. A NOESY spectrum of the amide proton–a-proton region of 1 mM peptide 109-141 in 20 mM sodium acetate with 100 mM deuterated SDS at pH 3.7 and 25°C. The sample was prepared by dispersing the peptide and the SDS into an aqueous solution with 50% HFIP, and the sample was then lyophilized and rehydrated. The mixing time used was 250 ms.

can be approximately interpreted as that the weaker the NOE cross-peak, the longer the corresponding interproton distance, and vice versa. For an a-helical structure, NOE connectivities corresponding to NHi − ai−3 and ai − bi+3 distances are expected to be ˚ , where i is the number of any residue in a E5 A protein or a peptide sequence (Wu¨thrich, 1986). We used all three criteria (chemical shift changes in comparison to random coil values, chemical shift indices, and NOE connectivities) to help identify secondary structures in peptides 109-122, 109-131 and 90-145. The results are shown in Figures 6 and 7. The chemical shift changes relative to the random coil values (Wishart et al., 1995) were corrected for nearest-neighbor effects and therefore may not

correspond exactly with the chemical shift indices based on the random coil values which were not corrected (Wishart & Sykes, 1994). Nevertheless, both of these criteria present the same overall features of the secondary structure. It is known that the residues at the N and C termini of a-helices in peptides and proteins are more conformationally mobile than the a-helical cores, as exemplified by the NMR studies of the trp repressor from Escherichia coli (Zhang et al., 1994). Thus, the chemical shift data and the NOE connectivities of the terminal residues need to be interpreted with caution. As shown in Figure 7, the chemical shifts for Val121 and Val122 of peptide 109-122 are distinct from those of peptide 109-141, and so are His140 and Phe141 of peptide 109-141

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Table 1. Chemical shift values (p.p.m.) of PrP peptide 109-141 a

b

b'

g

g'

4.19 4.22 4.76 4.44 4.25 3.91 4.17 4.18 4.18 4.08 3.83/3.91 4.25 3.91 3.84 3.94 4.07/3.92 4.34 3.85/3.97 3.83/3.91 4.40 4.34 4.20 3.91/3.96 4.37 4.27 4.42 4.48 4.50 4.39 4.40 4.30 4.55 4.54

2.16 1.72 3.18 2.05 1.43

2.24

2.61 1.36

2.65 1.41

3.37 2.13

2.52

2.57

0.93 0.95

1.04 1.03

Amino acid residue 109Met 110Lys 111His 112Met 113Ala 114Gly 115Ala 116Ala 117Ala 118Ala 119Gly 120Ala 121Val 122Val 123Gly 124Gly 125Leu 126Gly 127Gly 128Tyr 129Met 130Leu 131Gly 132Ser 133Ala 134Met 135Ser 136Arg 137Pro 138Met 139Met 140His 141Phe

d

d'

NH 8.36 8.36 8.26 8.16 8.23 8.23 7.98 8.09 7.99 8.05 8.26 7.84 7.81 7.97 8.17 7.97 8.03 8.33 8.12 7.83 8.12 8.02 8.19 7.90 8.19 7.93 7.90 8.00

1.68

1.45 1.45 1.45 1.45 1.49 2.24 2.17 1.84

1.68

1.70

3.07 2.08 1.71

2.52 1.61

2.60

2.55

2.63

1.70 1.94 2.54 2.44

1.73 1.99 2.62 2.52

3.87 1.43 2.04 3.81 1.83 2.30 2.08 1.94 3.05 2.93

0.89

0.89

0.93

0.91

3.90 2.10 3.87 1.85 2.10 1.95

3.21 3.83

8.09 7.83 7.97 7.83

3.21

Other 2.99 (e) 8.25 (H2 ), 7.45 (H4 ) 2.16 (e)

7.11 (H2,6 ), 6.79 (H3,5 )

2.06 (e) 7.19 (eNH)

6.92 (H4 ) 7.29 (H2,6 ), 7.31 (H4 ) 7.42 (H3,5 )

The peptide was dispersed in 20 mM sodium acetate with 100 mM SDS at pH 3.7 and 25°C.

from those of peptide 90-145. These results suggest that two to three residues from each terminus may exhibit conformational heterogeneity under our conditions and thereby may have adopted chemical shift values that are difficult to interpret. Although residues 109 and 110 in peptides 109-122 and 109-141 and residues 143, 144 and 145 in peptide 90-145

(a)

exhibit a-helical chemical shift values, they are probably affected by terminal flexibility and cannot be characterized confidently as a-helical. For peptide 90-145, chemical shift changes and chemical shift indices are shown only for residues 111 to 145 due to the absence of sufficient resonance connectivities for the other residues. Without these data, reliable

(b)

Figure 6. The NOE connectivities of (a) peptide 109-122 and (b) 109-141 in 20 mM sodium acetate with 100 mM SDS at pH 3.7 and 25°C. Broken lines represent overlapping connectivities. The putative a-helices H1 and H2 (Gasset et al., 1992; Huang et al., 1994) are underlined.

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(a)

(b)

(c)

assignments and a meaningful structural characterization are not possible for residues 90 to 110. Overall, residues 113 to 122 exhibited a-helical structures under our conditions as indicated by all three criteria. His111/Met112 displayed chemical shifts with random coil/b-sheet characteristics,

Figure 7. a-Proton chemical shift changes in comparison to random coil values and chemical shift indices (Wishart et al., 1992, 1995; Wishart & Sykes, 1994) for (a) peptide 109-122, (b) peptide 109-141, and (c) peptide 90-145 in 20 mM sodium acetate with 100 mM SDS at pH 3.7 and 25°C. The chemical shift values of nondegenerate Gly a-proton pairs were averaged. *Represents a missing assignment.

suggesting a possible b-break in the a-helical structure at this position. Arg136 was corrected in its chemical shifts for the effect of the neighboring proline (Wishart et al., 1995), and its index indicated characteristics of a b-turn. In addition, Pro137 did not exhibit any a-helical propensity as judged by its

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NOE connectivities. These are consistent with a possible break of the a-helical structure around Pro137. The few a-helical NOE connectivities and the sporadic a-helical chemical shifts as exhibited by residues 128 to 139 are consistent with a transient a-helix. Although Gly123 to Gly127 exhibited a-helical NOE connectivities (Figure 6(b)), their chemical shift values (Figure 7(b)) suggested characteristics of random coil or b-sheet. These seemingly contradicting results may be explained by the conformation of a reverse turn in which residue i to i + 3 (or i − 3) distances are close enough to create the corresponding NOE connectivities. The lack of long-range NOEs between H1 and H2 may be attributed to the flexible motion of H2 under our conditions. If such a reverse turn does indeed exist, it may suggest that the H1 helix plays a direct role in stabilizing the H2 helix. Formation of b-sheets Peptides 90-145 and 109-141 dissolved in aqueous solutions at physiological salt concentrations (100 to 140 mM sodium chloride) were studied by FTIR spectroscopy and were found to form progressively more intermolecular b-structures at pH 5 than at pH 7.2 (data not shown). A similar finding was reported for peptide 106-126 (Selvaggini et al., 1993). Judged by the gradual increase in the intensity of the absorption at 1623 cm−1 (Gasset et al., 1993), the b-sheet structure of peptide 90-145 at pH 5 was found to form during a period of a few days to a month (see Figure 8(a)). In the presence of acetonitrile, the time required for the b-sheet formation detectable by FTIR was found to decrease substantially at both pH 5 and 7.2, from approximately ten days to less than one day. As shown in Figure 8(b), FTIR absorption corresponding to intermolecular b-sheet structures was observed within 24 hours and continued to grow in intensity for two to three days. The b-sheet absorption was weaker in the absence of sodium chloride, suggesting that physiological salt concentrations were important in promoting b-sheet formation. The b-structures were found to exhibit rod-shaped aggregates in solutions at physiological sodium chloride concentration, with or without acetonitrile. As shown in Figure 9, peptide 90-145 in the b-sheet conformation exhibited rod-shaped polymers with diameters of 10 to 20 nm and variable length. This finding suggested that the b-sheet structures were stabilized by intermolecular interactions. Proteinase K resistance and epitope recognition The b-sheet forms of peptides 90-145 and 109-141 were found to be partially resistant to proteinase K digestion, while most of the corresponding non-b-sheet forms were completely degraded (see Figure 10). After the proteinase K digestion, peptide 109-141 retained its size while peptide 90-145 was reduced to a size similar to that of peptide 109-141 (Figure 10). This is analogous to the proteinase K

(a)

(b) Figure 8. FTIR spectra of peptide 90-145 upon the dispersion of the peptide in the solutions composed of (a) 20 mM sodium acetate with 140 mM sodium chloride at pH 5 and (b) 20 mM sodium acetate at pH 5 with 50% acetonitrile with (broken line) or without (continuous line) 140 mM sodium chloride on day 4. The vertical axis represents relative absorbance.

digestion of PrPSc (Oesch et al., 1985; Pan et al., 1993) where PrPSc was found to resist proteinase digestion, forming the non-digestible b-core, PrP 27-30. In our case, the proteinase-resistant b-core was formed by residues 109 to 141. As shown in Figure 11, antibodies 3F4 (mapping residues 108 and 111) and 13A5 (mapping residues 138 and 141) as well as antiserum RO73 (epitopes unknown) recognized peptide 90-145 in the b-sheet form before proteinase K digestion, but no antibody was able to recognize the peptide after the digestion. In addition, RO73 could not recognize peptide 109-141 either before or after digestion (results not shown). These findings argue, in addition to the

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Figure 9. Ultrastructure of peptide 90-145 upon the dispersion of the peptide in the solutions composed of; A, 20 mM sodium acetate at pH 5 on day 1; B, in 20 mM sodium acetate with 140 mM sodium chloride at pH 5 on day 17; and C, in 20 mM sodium acetate with 50% acetonitrile at pH 5 on day 4. The bar represents 100 nm.

evidence presented by the study of proteinase K digestion, that the amino acid sequence responsible for the b-sheet formation is likely to lie between residues 109 and 141. It appears that at least one of the epitopes recognized by the PrP antiserum RO73 lies within residues 90 to 108.

Discussion It is well established that the prion diseases are related to the post-translational conversion of PrPC to PrPSc (Borchelt et al., 1990; Prusiner, 1992). Several lines of evidence argue that this conversion involves a conformational transition in the prion

Figure 10. Coomassie blue-stained gel of peptides 109-141 (lanes 1 to 3) and 90-145 (lanes 4 to 6). Lanes 1 and 4, non-b-sheet form before proteinase K digestion; lanes 2 and 5, non-b-sheet form after proteinase K digestion; lanes 3 and 6, b-sheet form after proteinase K digestion. Molecular mass markers are shown on both sides of the gel in the order of (from top to bottom) 35 kDa, 28 kDa, 21 kDa and 7.2 kDa.

protein from a mostly a-helical structure (PrPC ) to a mostly b-sheet structure (PrPSc ) (Gasset et al., 1992; Pan et al., 1993; Cohen et al., 1994; Huang et al., 1994). To investigate the structural basis for the transition of PrPC to PrPSc, we studied peptides corresponding to some of the most evolutionarily conserved PrP residues (Scha¨tzl et al., 1995) and found that they can be converted to either a-helical or b-sheet structures under appropriate solution conditions. The a-helical structures are consistent with the proposed PrPC model (Gasset et al., 1992; Huang et al., 1994) while the residues that form the b-sheet structures in the PrP fragments overlap with those that adopt the a-helical conformation.

Figure 11. Immunoblotting of peptide 90-145 in b-sheet form before (lanes 1, 3 and 5) and after (lanes 2, 4 and 6) proteinase K digestion. Antibodies 3F4 (lanes 1 and 2) and 13A5 (lanes 3 and 4) as well as antiserum RO73 (lanes 5 and 6) were used for recognition. Molecular mass markers are shown on both sides of the gel in the order of (from top to bottom) 35 kDa, 28 kDa, 21 kDa and 7.2 kDa.

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Conformational Transitions of PrP Peptides

The a-helical structures Our findings suggest that hydrophobic interactions are responsible for the formation and intermolecular aggregation of the a-helical structures in the peptides containing the first two structured regions. This is in agreement with the proposal in the PrPC model (Huang et al., 1994) that the core formed by the four putative helices and the outer surfaces of the first two helices would be hydrophobic. We surmise that, in order for the PrPC to function as a cell-surface protein supported by a-helical folding (Borchelt et al., 1990; Pan et al., 1993; Huang et al., 1994), the first two a-helices must be protected from the hydrophilic extracellular milieu by packing with the remainder of the PrPC molecule. Because of the hydrophobic nature of the outer surfaces of the first two a-helices (Huang et al., 1994), a second PrPC molecule may also be needed to complete the hydrophobic packing. Whether the hydrophobic outer surfaces of the PrPC play a role in facilitating the PrPC-PrPSc heterodimer formation, which was proposed to be responsible for the production of PrPSc (Prusiner et al., 1990), remains to be determined. The NMR studies reported here indicate that residues 113 to 122 and 128 to 139 possess a-helical characteristics in SDS micelles. This finding is consistent with species variations in the PrP gene which tend to lie outside these regions (Scha¨tzl et al., 1995). The experimentally determined a-helical content is also consistent with that predicted by the modeling of PrPC (Gasset et al., 1992; Huang et al., 1994) where residues 109 to 122 and 129 to 141 were proposed to form a-helices. The partial a-helical characteristics of residues 128 to 139 in our peptides, as demonstrated by a few a-helical NOE connectivities and sporadic a-helical chemical shift values, may be attributed to dynamic averaging between two (or more) structural forms, i.e. between the random coil and one or more of the a-helical forms. Under such dynamic exchanges, we can observe only one averaged set of chemical shift values and weakened or absent NOE connectivities. Considering that only fragments of the PrP were examined, the structural instability may be attributed to the absence of structural support for folding from the remainder of the protein molecule. Under our conditions, residues 128 to 139 exhibit at least transient a-helical structures. The discrepancy between our experimental result and the prediction (Gasset et al., 1992; Huang et al., 1994) may be attributed to the following factors: (1) the prediction was based on a family of homologous sequences with the consideration of gene variations among species, whereas only the SHa PrP sequence was used in this study; (2) although hydrophobicity is required for the a-helical folding of the peptide fragments, the hydrophobic interior of the SDS micelles may not mimic the exact folding environment of the peptide fragments in PrPC; and (3) the precision of the homology-based prediction protocols may need further refinement. The CD data indicated that putative H2 in isolation

could not assume an a-helical structure in solution. It could, however, be induced to form a partial a-helical structure in the presence of H1, as is evident in our NMR data. Similarly, the H1 region was shown to be stabilized by the H2 region, as was shown in our data that peptide 109-141 exhibited larger upfield chemical shift changes and more extensive a-helical connectivities within the H1 region than does peptide 109-122 alone (see Figure 7). It is likely that such cooperative folding may be further enhanced in the native protein (PrPC ) by the interactive packing of all four helices. Whether the cooperativity is disrupted by point mutations of the human PRNP gene which segregate with human GSS diseases (Prusiner & Hsiao, 1994) remains to be established. The b-sheet structures The resistance of the peptides to proteinase K digestion in the b-sheet form established a correlation between the peptides and the PrP isoform, PrPSc (Oesch et al., 1985). Residues 109 to 141 were shown to be capable of forming a proteinase-resistant core which formed fibrils analogous to PrP 27-30 (Pan et al., 1993). Similar amyloid-like fibrils were observed in two smaller fragments of the first two helical regions in an attempt to study the fibril deposition of the human GSS disease with a Phe to Ser mutation at PRNP codon 198 (Tagliavini et al., 1993). Although the precise N and C termini of the b-structures cannot be determined by this study, recent solid-state NMR (A. C. Kolbert et al., unpublished results) and FTIR results (M. A. Baldwin et al., unpublished results) on SHa PrP peptide 109-122 and other related peptides suggest that the b-structures begin near Ala113. Our finding that the same region in peptides 90-145 and 109-141 formed both a-helical and b-sheet structures is consistent with the proposal that the PrP may adopt more than one energetically favorable conformation (Cohen et al., 1994; Huang et al., 1994). What PrP conformation exists in the cell is likely to depend on the environment in which the PrP molecule folds. Our results raise the possibility that several b-sheet conformations of PrP may possess comparably favorable folding energies (Cohen et al., 1994) and that these conformations might be manifest as multiple prion ‘‘strains’’. The molecular basis of prion strains remains enigmatic but these isolates are distinguished by their incubation times, distribution of vacuolar lesions and patterns of PrPSc accumulation (Dickinson et al., 1968; Bruce & Dickinson, 1987; Hecker et al., 1992). The proteinase-resistant core found in this study encompasses some of the most-conserved residues of the PrP (Scha¨tzl et al., 1995). Several lines of evidence as presented in this study and other studies (Gasset et al., 1992; Selvaggini et al., 1993; Tagliavini et al., 1993; Nguyen et al., 1995) suggest that the b-core-forming residues 109 to 141, especially residues 113 to 122, are the most amyloidogenic. Whether the fragments containing the b-core can be converted into an infectious form in vitro remains to

JMB—MS 634 524 be established. It has been reported recently that PrPC can be converted in vitro to proteinase-resistant forms in the presence of PrPSc (Kocisko et al., 1994). These findings demand further verification since the 50-fold excess PrPSc used in the conversion excluded the possibility to measure the formation of nascent infectivity and to determine the conformation of the ‘‘converted PrP’’. Our study showed that residues His111-Met112 and the residues adjacent to Pro137 exhibited weak or no a-helical propensity under our a-helix-forming conditions. Interestingly, computational studies suggest that the residues in the same regions could form b-hairpins in a model of PrPSc (Z. Huang et al., unpublished results). We therefore surmise that these residues may be important in the conversion of PrPC to PrPSc where the prion protein changes its conformation from a mostly a-helical structure to one that is rich in b-sheet structure (Gasset et al., 1992; Pan et al., 1993; Cohen et al., 1994; Huang et al., 1994). It is possible that this relatively weak a-helix-forming (or stronger b-forming) propensity of the residues adjacent to His111-Met112 and Pro137 in comparison to the rest of the sequence may render PrPC intrinsically susceptible to the structural conversion following a perturbation in the hydrophobic folding environment. Salt at physiological concentrations was found to be important in promoting intermolecular b-structures in PrP peptides. It may also be important for inducing structural changes in PrPC. We expect that the normal folding of PrPC would protect the hydrophobic interior of the molecule from the hydrophilic aqueous environment. However, mutations that destabilize the hydrophobic packing, such as germline mutations in the inherited prion diseases or postulated somatic mutations as may occur in sporadic prion diseases (Prusiner, 1991) could expose the hydrophobic core of the protein to the extracellular milieu. Prolonged exposure to this milieu might provoke mutant PrPC to be converted to PrPSc instead of being hydrolyzed to a 17 kDa polypeptide within a non-acidic, cholesterol-rich microdomain (Taraboulos et al., 1995). Alternatively, a particular microenvironment may alter the conformation of wild-type PrPC in such a way that it spontaneously refolds into PrPSc. Both somatic mutations of the PrP gene and the spontaneous conversion of PrPC into PrPSc have been postulated to explain sporadic CJD, which is the most common form of human prion disease (Prusiner, 1991).

Materials and Methods Peptide preparation Peptides 109-141 and 90-145, as well as 104-122, 109-122 and 129-141 were synthesized from amino acid residues protected by 9-fluorenylmethoxycarbonyl (Fmoc) on a Millipore (Bedford, MA) model 9050 Plus PepSynthesizer. Thioanisole/trifluoroacetic acid/water or phenol/trifluoroacetic acid/water was used for cleavage. The N termini of the peptides were free of acetylation, and the

Conformational Transitions of PrP Peptides

C termini of the peptides were amidated. The peptides were purified by reverse phase high performance liquid chromatography (HPLC). To remove residual trifluoroacetic acid, three-times excess of hydrochloric acid was added to the collected fractions. The fractions were lyophilized, suspended in 50% (v/v) acetonitrile/water and relyophilized. Fourier transform infrared spectroscopy (FTIR) Peptides were dissolved in 20 mM sodium acetate/2H2O at pH 3.7 or 5, with 0 mM to 140 mM NaCl, or 10 to 50% (v/v) of an organic solvent (ACN, TFE or HFIP), or 100 mM detergent (SDS or DPC). Peptide concentrations were between 1 and 1.5 mM. FTIR spectra were collected on a Perkin–Elmer System 2000 spectrophotometer equipped with a microscope continuously purged with nitrogen. A 0.05 mm path-length cell with barium fluoride windows was used with the microscope. Spectra of 100 scans each were accumulated for both the peptide solutions and the buffers under identical conditions. The final spectra were obtained by subtraction of the buffer spectra. Circular dichroism spectroscopy (CD) Peptide solutions were prepared as for the FTIR studies except that the peptide concentrations were between 0.5 and 1 mM. CD spectra were collected at 25°C on a Jasco model 720 spectropolarimeter equipped with a stress-plate modulator and continuously purged by dry nitrogen. Calibration was carried out using an aqueous solution of (+)-10-camphorsulfonic acid. Spectra of four to ten scans each were accumulated using a 0.01 or 0.02 cm path-length cell for both the peptide solutions and the buffer under identical conditions. The final spectra were obtained by subtraction of the buffer spectra. Mean residue molar ellipticity was calculated based on the peptide concentration, the number of residues in the peptide and the cell path-length.

Electron microscopy (EM) Peptide solutions were prepared as described in the CD and FTIR studies and diluted to about 0.5 to 2 mg/ml. Pioliform resin (0.8%) and carbon-coated grids (400 mesh) were floated on drops of the diluted peptide solutions before fixing by vapor diffusion of 50% (v/v) glutaraldehyde for fixation. The grids were then negatively stained with 1% (w/v) phosphotungstic acid. A JEOL 100CX electron microscope operated at 80 keV was used to collect electron micrographs. NMR spectroscopy NMR samples were prepared by dissolving the peptides in 20 mM deuterated sodium acetate at pH 3.7 and 5, with 10 to 30% (v/v) deuterated HFIP or 100 mM deuterated SDS (Cambridge Isotope Laboratories, Andover, MA). The samples at pH 7.2 were prepared using 20 mM potassium phosphate instead of sodium acetate. The peptide concentrations were 0.5 to 1 mM. All spectra were obtained on a Varian 600 MHz UnityPlus spectrometer equipped with a 10 mm single channel probe. The temperatures used were between 5 and 35°C and spectral width 7000 Hz. For two-dimensional spectra such as nuclear Overhauser enhancement spectroscopy (NOESY) and total correlated spectroscopy (TOCSY), 2048 data points were collected in the t2 dimension and 512 transients

JMB—MS 634 Conformational Transitions of PrP Peptides

in the t1 dimension. The spectra were processed with zero-filling of the t1 dimension to 2k and apodized by a sine-squared window function shifted by 60° in both dimensions. Mixing times used were 70 ms, 150 ms and 250 ms for NOESY, and 70 ms for TOCSY. All spectra were referenced to an internal standard of 0.24 mM 3-(trimethylsilyl)-tetradeutero-sodium propionate (TSP).

Proteinase K digestion and antibody recognition Preparation of the b-sheet forms of the peptides 109-141 and 90-145 was achieved by dispersing the peptides in 20 mM Hepes with 50% acetonitrile and 100 mM sodium chloride at pH 7.4. Samples were stored at 4°C for up to 30 days to allow the development of b-sheet. Proteinase K digestion was carried out on both b-sheet and non-b-sheet forms of the peptides (10 mg/ml) using 50 mg/ml proteinase K in 0.2 to 1% (w/v) Sarkosyl at pH 7.4 and 37°C. The digestion was stopped by incubation with 5 mM phenylmethylsulfonylfluoride (PMSF) at room temperature for ten minutes. These samples, as well as controls, were then separated on a SDS-15% PAGE gel as described (Laemmli, 1970) with 4 mg of one of the peptides per lane. The gel was stained with Coomassie blue and transferred to a Millipore Immobilon-P membrane. Immunoblotting of the peptides was carried out as described (Rogers et al., 1991) using antiserum RO73 and antibodies 3F4 and 13A5. RO73 is a rabbit polyclonal antiserum raised against electrophoretically purified PrP 27-30 with unknown epitope pattern. 3F4 and 13A5 are mouse monoclonal antibodies. which were shown to recognize amino residues 108 and 111, and 138 and 141, respectively, of the SHa PrP proteins (Rogers et al., 1991).

Acknowledgements This work was supported by NIH grants and a grant from the American Health Assistance Foundation, as well as by gifts from Sherman Fairchild Foundation and Bernard Osher Foundation.

References Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N. & Prusiner, S. B. (1990). Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J. Cell Biol. 110, 743–752. Brown, P., Gibbs, C. J., Jr, Rodgers-Johnson, P., Asher, D. M., Sulima, M. P., Bacote, A., Goldfarb, L. G. & Gajdusek, D. C. (1994). Human spongiform encephalopathy: the National Institutes of Health series of 300 cases of experimentally transmitted disease. Ann. Neurol. 35, 513–529. Bruce, M. E. & Dickinson, A. G. (1987). Biological evidence that the scrapie agent has an independent genome. J. Gen. Virol. 68, 79–89. Caughey, B. W., Dong, A., Bhat, K. S., Ernst, D., Hayes, S. F. & Caughey, W. S. (1991). Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry, 30, 7672–7680. Cohen, F. E., Pan, K.-M., Huang, Z., Baldwin, M., Fletterick, R. J. & Prusiner, S. B. (1994). Structural clues to prion replication. Science, 264, 530–531.

525 Dickinson, A. G., Meikle, V. M. H. & Fraser, H. (1968). Identification of a gene which controls the incubation period of some strains of scrapie agent in mice. J. Comp. Pathol. 78, 293–299. Gajdusek, D. C. (1977). Unconventional viruses and the origin and disappearance of kuru. Science, 197, 943–960. Gasset, M., Baldwin, M. A., Lloyd, D., Gabriel, J.-M., Holtzman, D. M., Cohen, F., 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. A., Fletterick, R. J. & Prusiner, S. B. (1993). Perturbation of the secondary structure of the scrapie prion protein under conditions associated with changes in infectivity. Proc. Natl Acad. Sci. USA, 90, 1–5. Hecker, R., Taraboulos, A., Scott, M., Pan, K.-M., Torchia, M., Jendroska, K., DeArmond, S. J. & Prusiner, S. B. (1992). Replication of distinct prion isolates is region specific in brains of transgenic mice and hamsters. Genes Dev. 6, 1213–1228. Huang, Z., Gabriel, J.-M., Baldwin, M. A., Fletterick, R. J., Prusiner, S. B. & Cohen, F. E. (1994). Proposed three-dimensional structure for the cellular prion protein. Proc. Natl Acad. Sci. USA, 91, 7139–7143. Kitamoto, T., Iizuka, R. & Tateishi, J. (1993). An amber mutation of prion protein in Gerstmann-Stra¨ussler syndrome with mutant PrP plaques. Biochem. Biophys. Res. Commun. 192, 525–531. Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. T., Jr & Caughey, B. (1994). Cell-free formation of protease-resistant prion protein. Nature, 370, 471–474. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature, 227, 680–685. Merutka, G., Dyson, H. J. & Wright, P. E. (1995). ‘Random coil’ 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG. J. Biomol. NMR, 5, 14–24. Meyer, R. K., McKinley, M. P., Bowman, K. A., Braunfeld, M. B., Barry, R. A. & Prusiner, S. B. (1986). Separation and properties of cellular and scrapie prion proteins. Proc. Natl Acad. Sci. USA, 83, 2310–2314. Nguyen, J., Baldwin, M. A., Cohen, F. E. & Prusiner, S. B. (1995). Prion protein peptides induce a-helix to b-sheet conformational transitions. Biochemistry, 34, 4186–4192. Oesch, B., Westaway, D., Wa¨lchli, M., McKinley, M. P., Kent, S. B. H., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B., Hood, L. E., Prusiner, S. B. & Weissmann, C. (1985). A cellular gene encodes scrapie PrP 27-30 protein. Cell, 40, 735–746. Pan, K.-M., Baldwin, M., Nguyen, J., Gasset, 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, 10962–10966. Prusiner, S. B. (1991). Molecular biology of prion diseases. Science, 252, 1515–1522. Prusiner, S. B. (1992). Chemistry and biology of prions. Biochemistry, 31, 12278–12288. Prusiner, S. B. (1994a). Biology and genetics of prion diseases. Annu. Rev. Microbiol. 48, 655–686. Prusiner, S. B. (1994b). Inherited prion diseases. Proc. Natl Acad. Sci. USA, 91, 4611–4614.

JMB—MS 634 526 Prusiner, S. B. & Hsiao, K. K. (1994). Human prion diseases. Ann. Neurol. 35, 385–395. Prusiner, S. B., Bolton, D. C., Groth, D. F., Bowman, K. A., Cochran, S. P. & McKinley, M. P. (1982). Further purification and characterization of scrapie prions. Biochemistry, 21, 6942–6950. Prusiner, S. B., McKinley, M. P., Bowman, K. A., Bolton, D. C., Bendheim, P. E., Groth, D. F. & Glenner, G. G. (1983). Scrapie prions aggregate to form amyloid-like birefringent rods. Cell, 35, 349–358. Prusiner, S. B., Groth, D. F., Bolton, D. C., Kent, S. B. & Hood, L. E. (1984). Purification and structural studies of a major scrapie prion protein. Cell, 38, 127–134. Prusiner, S. B., Scott, M., Foster, D., Pan, K.-M., Groth, D., Mirenda, C., Torchia, M., Yang, S.-L., Serban, D., Carlson, G. A., Hoppe, P. C., Westaway, D. & DeArmond, S. J. (1990). Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell, 63, 673–686. Rogers, M., Serban D., Gyuris, T., Scott, M., Torchia, T. & Prusiner, S. B. (1991). Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system. J. Immunol. 147, 3568–3574. Safar, J., Roller, P. P., Gajdusek, D. C. & Gibbs, C. J., Jr (1993). Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein. J. Biol. Chem. 268, 20276–20284. Scha¨tzl, H. M., Da Costa, M., Taylor, L., Cohen, F. E. & Prusiner, S. B. (1995). Prion protein gene variation among primates. J. Mol. Biol. 245, 362–374. Selvaggini, C., De Gioia, L., Cantu, L., Ghibaudi, E., Diomede, L., Passerini, F., Forloni, G., Bugiani, O., Tagliavini, F. & Salmona, M. (1993). Molecular characteristics of a protease resistant, amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein. Biochem. Biophys. Res. Commun. 194, 1380–1386. Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L. & Prusiner, S. B. (1993). Structural analysis of the scrapie prion protein using

Conformational Transitions of PrP Peptides

mass spectrometry and amino acid sequencing. Biochemistry, 32, 1991–2002. Tagliavini, F., Prelli, F., Verga, L., Giaccone, G., Sarma, 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 106-147 form amyloid-like fibrils in vitro. Proc. Natl Acad. Sci. USA, 90, 9678–9682. Taraboulos, A., Scott, M., Semenov, A., Avrahami, D., Laszlo, L. & Prusiner, S. B. (1995). Cholesterol depletion and modification of C-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform. J. Cell Biol. 129, 121–132. Tateishi, J., Doh-ura, K., Kitamoto, T., Tranchant, C., Steinmetz, G., Warter, J. M. & Boellaard, J. W. (1992). Prion protein gene analysis and transmission studies of Creutzfeldt-Jakob disease. In Prion Diseases of Humans and Animals (Prusiner, S. B., Collinge, J., Powell, J. & Anderton, B., eds), pp. 129–134, Ellis Horwood, London. Wilesmith, J. W. (1994). Bovine spongiform encephalopathy: epidemiological factors associated with the emergence of an important new animal pathogen in Great Britain. Semin. Virol. 5, 179–187. Wishart, D. S. & Sykes, B. D. (1994). Chemical shifts as a tool for structure determination. Methods Enzymol. 239, 363–393. Wishart, D. S., Sykes, B. D. & Richards, F. M. (1992). Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. Biochemistry, 31, 1647–1651. Wishart, D. S., Bigam, C. G., Holm, A., Hodges, R. S. & Sykes B. D. (1995). 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J. Biomol. NMR, 5, 67–81. Wu¨thrich, K. (1986). NMR of Proteins and Nucleic Acids, New York, Wiley-Interscience. Zhang, H., Zhao, D., Revington, M., Lee, W., Jia, X., Arrowsmith, C. & Jardetzky, O. (1994). The solution structures of the trp repressor-operator DNA complex. J. Mol. Biol. 238, 592–614.

Edited by A. Fersht (Received 3 March 1995; accepted 28 April 1995)