A backbone-reversed all-ß polypeptide (retro-CspA) folds and assembles into amyloid nanofibres

Anshuman Shukla, Manoj Raje and Purnananda Guptasarma1

Institute of Microbial Technology, Sector 39-A, Chandigarh, India

1 To whom correspondence should be addressed. e-mail: pg{at}imtech.res.in


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The backbone-reversed or ‘retro’, form of a model all-ß-sheet protein, Escherichia coli CspA, was produced from a synthetic gene in E.coli in fusion with an N-terminal affinity tag. Following purification under denaturing conditions and dialysis-based removal of urea, the protein was found to fold into a soluble, poorly structured multimer. Upon concentration, this state readily transformed into amyloid nanofibres. Congo Red-binding amorphous forms were also observed. Since a ß-sheet-forming sequence is expected to retain high ß-sheet-forming propensity even after backbone reversal and given the fact that folding of retro-CspA occurs only to a poorly structured form, we conclude that the increase effected in protein concentration may be responsible for the formation of intermolecular ß-sheets, facilitating the bleeding away of the protein’s conformational equilibrium into aggregates that generate well-formed fibres. Since every molecule in these fibres contains a peptide tag for binding Ni2+, the fibres may provide a template for deposition of nickel to generate novel materials.

Keywords: amyloid fibre formation/backbone-reversal/protein aggregation/protein misfolding/retro modification


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Amyloid fibres are constituted of polypeptides that assemble through the formation of intermolecular ß-sheets (Sunde et al., 1997Go). It may be reasonably argued, therefore, that amyloids should be formed relatively easily by any polypeptide that (i) displays a propensity to form ß-sheets, while (ii) being somehow compromised in respect of the ability to fold rapidly to a stable ß-sheet structure(s) through a unimolecular folding reaction.

To test this contention, we chose for extensive sequence modification a 70 residues long all-ß-sheet protein, the cold shock protein A (CspA) of Escherichia coli (Reid et al., 1998Go). The amino acid sequence of this all-ß-sheet protein was reversed by molecular genetic methods to create a novel protein, retro-CspA, through the synthesis, transformation and expression in E.coli of a synthetic gene encoding the backbone-reversed sequence of CspA in fusion with an N-terminal 6xHis affinity tag. The rationale for making this ‘retro’ modification was as follows: sequence reversal can be expected to cause no change in a polypeptide’s propensity to adopt a ß structure, since neither the residues themselves (with their individual ß-sheet-forming propensities) nor their relative groupings in the chain tend to be affected by the modification. Sequence reversal could, however, potentially compromise a polypeptide’s ability to fold into a stable structure. Thus, for instance, even if the ‘retro’ form of an all-ß-sheet protein were to display the ability to fold to a structure constituting a topological mirror image of that adopted by the parent sequence [as has been proposed previously (Guptasarma, 1992Go, 1996Go)], it is likely that folded forms of such a protein would co-exist in equilibrium with partially folded and unfolded forms. This would be particularly so if folding failed to convert chains into sufficiently thermodynamically stabilized structures.

At high protein concentrations, partially folded forms of a retro-all-ß-sheet protein could thus conceivably associate through the formation of intermolecular ß-sheets into proto-amyloid structures that could then be expected to bleed progressively a fragile conformational equilibrium in solution away towards the aggregated state. Here we show that at low protein concentrations (<~1 mg/ml) retro-CspA adopts a structured, multimeric form, but that upon raising the protein concentration or upon extended incubation, retro-CspA transforms readily into amorphous and fibril-like amyloid structures.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Gene synthesis

Contract synthesis of double-stranded DNA segments was carried out by Gemini Biotech (USA). Further modifications, additions of sequences, mutations and cloning were carried out using standard molecular biological procedures in the laboratory.

Forms of retro-CspA constructed

Using the same codons as used naturally by the gene encoding CspA in the E.coli genome, but through appropriate shuffling of codons to create a new structural gene incorporating the codons in the reversed sequence, we created a construct (RETCSPA-1) encoding retro-CspA in fusion with a 14 residues long N-terminal affinity tag cloned between the restriction sites for SphI and SalI in the vector pQE-30 from Qiagen; in addition, this construct also possessed a nine residues long C-terminal extension owing to the cloning site. The N-terminal affinity tag, MRGSHHHHHHGSAC, incorporated an RGS sequence along with six consecutive histidine residues and four other residues contributed by the vector’s cloning site. The C-terminal extension derived from the cloning site had the sequence VDLQPSLIS. Together with its extensions, the polypeptide encoded by the construct RETCSPA-1 was 93 residues long. Subsequently another form of the protein, RETCSPA-2, was also created; this form lacked the C-terminal extension and also the last two residues of the N-terminal tag, causing it to be only 82 residues long. DNA sequencing was carried out to confirm the integrity of the construct. The amino acid sequences of the two retro-CspA constructs are shown in Figure 1 together with the sequence of the parent protein, CspA. Transformation into E.coli M15pREP4 led to expression of both forms with yields of ~15 mg/l of culture. The two protein constructs were purified under denaturing conditions using standard protocols (Qiagen) for Ni-NTA affinity chromatography, through lysis of cells in 8 M urea. Following purification in the presence of urea, the proteins were allowed to fold by dialyzing out the urea. N-terminal sequencing of the first 10 residues was used to confirm the identities of the purified proteins.



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Fig. 1. Sequences of CspA of E.coli and the sequences of the two retroprotein constructs studied, RETCSP-1 and RETCSP-2.

 
Solution-state and solid-state studies of samples

The proteins were found to be soluble at low concentrations (<1 mg/ml). Consequently, all solution studies were done with samples of concentrations <1 mg/ml. With increase in concentration, through vacuum centrifugation, to >~1.2 mg/ml, precipitates were observed to accumulate at the bottom of microcentrifuge tubes. At concentrations of 1.0–1.2 mg/ml, incubation of the supernatant through standing for a few tens of hours at 4°C also produced precipitates, for reasons detailed in the Results.

Spectroscopy

Protein concentrations were estimated through UV absorption measurements at 280 nm, using a predicted molar extinction coefficient of 6970 for proteins encoded by both RETCSP-1 and RETCSP-2, which are referred to hereafter by the same names. Fluorescence spectra were collected on a Perkin-Elmer LS-50B spectrofluorimeter using excitation and emission bandpasses of 5 nm each, using excitation with radiation of 280 nm and scanning protein emission between 300 and 400 nm. Circular dichroism (CD) spectra were collected at intervals of 1 nm on a Jasco J-810 spectropolarimeter through scanning of wavelengths from 250 to 200 nm, using a protein concentration of 0.4 mg/ml and a cuvette pathlength of 0.2 cm. CD signals below 200 nm could not be collected because the spectra were noisy; consequently, no attempt was made to estimate secondary structural contents from these data.

Chromatography

Gel filtration chromatography was performed on a Pharmacia SMART system using an analytical Superdex-200 column (bed volume ~2.4 ml; void volume 0.8 ml) and a flow rate of 0.1 ml/min, through use of 0.05 ml protein samples of concentration 0.4 mg/ml. The fractionation range of the column was 600 000 Da and the exclusion limit 1 600 000 Da.

Fluorescence microscopy

Proteins in the amyloid state are reported to bind the dye Congo Red with pink–red fluorescence observed from the bound dye upon excitation with UV radiation (Linke, 2000Go). We examined the Congo Red-binding ability of some amorphous forms of retro-CspA precipitates using fluorescence imaging on a Nikon Eclipse E-600 microscope. Excitation was carried out through a filter allowing light of 350–390 nm to fall on the sample. Precipitated protein was placed under a cover-slip on a slide and the dye allowed to diffuse in from the sides of the cover-slip. The titration of the dye by the protein was observed visually.

Visible microscopic examination of affinity-tag accessibility

The accessibility of the N-terminal 6xHis affinity tags on the RETCSP-1 polypeptide to molecules in the solvent was assessed qualitatively through binding of Ni-NTA–horseradish peroxidase conjugates. Visual examination was carried out with the fluorescence microscope in the ordinary optical mode, after carrying out a peroxide-based staining reaction of the protein using standard protocols (Qiagen) on a slide. For this, all reagents were premixed with the protein and diaminobenzamidine (DAB) was allowed to leach in from the sides of the cover-slip.

Transmission electron microscopy (TEM)

TEM studies were carried out through use of routine negative staining procedures employing PTA and uranyl acetate.

Amino acid sequencing

N-terminal sequences of the samples were determined through Edman degradation chemistry on an Applied Biosystems 476A protein sequencer,


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Evidence for structural collapse and association

The N-terminal sequences of the purified, overexpressed proteins were determined after blotting of the appropriate protein bands from SDS–PAGE and found to be MRGSHHHHHH, confirming their identities. The folding of RETCSP-1 and RETCSP-2 from the denatured state through dialysis of urea is discussed in the Materials and methods. Representative fluorescence spectra, CD spectra and a gel filtration chromatogram of the soluble form of RETCSPA-2 (the form of retro-CspA lacking the C-terminal extension; see Materials and methods) are shown in Figure 2, together with the calibration data for the analytical column used. The data obtained can be summarized as follows: the fluorescence emission spectrum of the protein (Figure 2a) shows a distinct maximum at ~342 nm, indicating partial burial of its lone tryptophan residue away from the aqueous solvent. As is well known, proteins that are completely unfolded emit maximally at 352–353 nm (Schmid, 1989Go).



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Fig. 2. Structural biochemical behavior of RETCSP-2. (a) Fluorescence emission spectrum; (b) CD spectra at 25°C (open symbols) and 4°C (closed symbols); (c) gel filtration elution chromatogram on a SMART analytical Superdex-200 column; (d) calibration data for the column shown in (c).

 
The CD spectrum of the protein at room temperature (Figure 2b) collected at a protein concentration of 0.4 mg/ml shows a negative band minimum at about 205 nm. That there is significant negative ellipticity at 210 nm indicates that the protein contains secondary structure, since for a perfectly randomly coiled protein there is no CD signal between 250 and 210 nm and negative ellipticity tends to be seen only below this range of wavelengths with a minimum at 198 nm. Notably, the CD spectrum of normal CspA also has a similar shape and a minimum at 205 nm (Reid et al., 1998Go); however, the intensity of the band minimum is more than three times that of retro-CspA and the signal strength at 210 nm, relative to that at 205 nm, is also higher with CspA than with RETCSP-2, indicating that some random coil structure (at least some of which could be due to the 12 residues long N-terminal extension, MRGSHHHHHHGSAC) is present in this folded form of retro-CspA. The CD spectrum of the protein at 4°C is also shown alongside in Figure 2b, from which it is evident that structural destabilization occurs with cooling. Such facile cold denaturation suggests that the structure formed cannot be significantly stable, since few naturally occurring proteins show cold denaturation above 0°C.

On a Superdex-200 gel filtration column, most of the RETCSP-2 population was found to elute at a volume of 1.05 ml (Figure 2c), suggesting that the polypeptides have associated to a molecular weight approaching that of apoferritin, which is about 443 kDa, and elutes at 1.07 ml from the same column. The calibration data are shown in Figure 2d.

With RETCSP-1, the wavelength of maximal fluorescence emission turned out to be the same as that characteristic of RETCSP-2, i.e. 342–343 nm. Similarly, RETCSP-1 also displayed a similar association state, as assessed by gel filtration chromatography, with most of the population eluting at 1.05 ml, indicative of the adoption of a size above 400 kDa. The far-UV CD spectrum of RETCSP-1 indicated a greater content of randomly coiled structure. This was presumably because of the extra C-terminal tag (absent in RETCSP-2), which may be expected to have remained unstructured. One reason to believe this to be the case is that the CD spectrum of RETCSP-1 also showed negative ellipticity at 210 nm comparable to that seen in RETCSP-2, indicating that the extra negative ellipticity at 200 nm was due to the randomly coiled structure of the C-terminal tag. Since these data for RETCSP-1 do not provide any additional insights, they are not shown separately. Importantly, with both forms, we failed to see any significant change in enthalpy upon heating in a differential scanning calorimeter (data not shown), supporting the likelihood of the adoption of a poorly structurally-stabilized form.

Collectively, therefore, the above pieces of evidence demonstrate that the soluble form of the protein at concentrations <~1 mg/ml is not that of an unfolded monomer unable to adopt secondary and tertiary structure. Rather, the protein clearly forms secondary structure, buries away its lone tryptophan residue (indicating side chain–side chain interactions and formation of some tertiary structure) and associates to form a very large multimeric agglomerate.

Light microscopic examination of the amyloid-like state

The precipitated form of the protein displays both amorphous and fibre-like morphologies. Both forms readily bind the dye Congo Red and cause it to display a red fluorescence upon illumination with UV radiation (Figure 3a–c) that has recently been reported to be characteristic of an amyloid-like ß structural organization at the molecular level (Linke, 2000Go). Furthermore, molecules in the protein precipitate appear to expose their N-terminal 6xHis affinity tags. The Ni-NTA conjugated form of the enzyme horseradish peroxidase binds to the tag in the precipitated state of the protein, to stain the region of the precipitate in which binding occurs (Figure 3d), reinforcing the likelihood of the N-terminal extension being unstructured. Since the data with RETCSP-1 and RETCSP-2 were similar, only data for the former construct are shown.



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Fig. 3. Typical light microscopic (ad) and transmission mode electron microscopic images (eg) of RETCSP-1 aggregates. (a) A representative amorphous aggregate of CspA titrating out Congo Red from solution (diffused in through the sides of the cover-slip). The image is one of an aggregate that has just begun to bind the dye at its periphery, seen as red fluorescence upon UV excitation. (b) A typical amorphous aggregate that has become completely stained by Congo Red. (c) Fibrillar aggregates stained by Congo Red. (d) Staining (brown) of amorphous aggregate material by Ni-NTA–horseradish peroxidase conjugate. (e–g) TEM images of fields of amyloid fibres at various magnifications.

 
Electron microscopic examination of the retro-CspA amyloid fibre

Figure 3e–g shows the morphology of the amyloid fibrils formed by RETCSP-1 in different fields and at different levels of magnification. A variety of fibril diameters are observed along with visual evidence of twisting of fibrils of smaller diameter to form fibres that are 70–80 nm in diameter, explaining how fibrils are also observed in ordinary fluorescence microscopy at high magnification (Figure 3c). Extensive formation of fibrils was also observed with RETCSP-2, with an apparently greater degree of intertwining and branching.

Conclusion

The work presented in this paper demonstrates that a polypeptide chain possessing a strong tendency to form ß-sheet structures may form amyloid-like (microstructured) amorphous aggregates in addition to amyloid fibrils if it is unable to fold into a sufficiently stable three-dimensional structure. It appears likely that any existence of poorly folded forms in conformational equilibrium with folded as well as unfolded forms could potentially facilitate the progressive formation of aggregated structures that are more kinetically (and possibly also thermodynamically) stable than either unfolded or folded forms. Once such aggregation starts, it could proceed through the bleeding away of the equilibrium by the aggregate since aggregation is a largely irreversible phenomenon. It must be emphasized that although retro-CspA behaves in this interesting manner and indeed any backbone-reversed ß-sheet protein that fails to fold to a stable structure could also be expected to form amyloids, we are not implying that all backbone-reversed ß-sheet proteins are likely to fail to fold. Indeed, two other short (10–12 kDa) backbone-reversed ß-sheet proteins produced in our laboratory, with identical N-terminal 6xHis tags, turn out to fold very well and do not behave like CspA (Shukla et al., 2003Go; A.Shukla and P.Guptasarma, submitted). Further studies with retro-proteins are in progress.


    Acknowledgements
 
A.S. thanks the Council of Scientific and Industrial Research, New Delhi, for a doctoral research fellowship. P.G. thanks the CSIR New Idea Fund, Indian National Science Academy, New Delhi, and TWAS, Trieste, for projects funding research into protein folding and aggregation. A.Theophilus is thanked for technical help with TEM.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Guptasarma,P. (1992) FEBS Lett., 310, 205–210.[CrossRef][ISI][Medline]

Guptasarma,P. (1996) Trends Biotechnol., 14, 42–43.[CrossRef][ISI]

Linke,R.P. (2000) Virchows Arch., 436, 439–448.[CrossRef][ISI][Medline]

Reid,K.L., Rodriguez,H.M., Hillier,B.J. and Gregoret,L.M. (1998) Protein Sci., 7, 470–479.[Abstract/Free Full Text]

Schmid,F.X. (1989) In Creighton,T.E. (ed.), Protein Structure: A Practical Approach. IRL Press, Oxford, pp. 251–285.

Shukla,A., Raje,M. and Guptasarma,P. (2003) J. Biol. Chem., 278, 26505–26510.[Abstract/Free Full Text]

Sunde,M., Serpell,L.C., Bartlam,M., Fraser,P.E. and Blake,C.C. (1997) J. Mol. Biol., 273, 729–739.[CrossRef][ISI][Medline]

Received April 1, 2003; revised October 27, 2003; accepted October 30, 2003





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