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Stability and conformational properties of doppel, a prion-like protein, and its single-disulphide mutant
Sheena M Whyte1, Ian D Sylvester, Stephen R Martin
1National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Abstract:
Both prion protein and the structurally homologous protein doppel are associated with neurodegenerative disease by mechanisms which remain elusive. We have prepared murine doppel, and a mutant with one of the two disulphide bonds removed, in the expectation of increasing the similarity of doppel to prion protein in terms of conformation and stability. Unfolding studies of doppel and the mutant have been performed using far-UV CD over a range of solution conditions known to favour the alpha-->beta transformation of recombinant prion protein. Only partial unfolding of doppel or the mutant occurs at elevated temperature, but both exhibit full and reversible unfolding in chemical denaturation with urea. Doppel is significantly less stable than prion protein, and this stability is further reduced by removal of the disulphide bond between residues 95-148. Both doppel and the mutant are observed to unfold by a two-state mechanism, even under the mildly acidic conditions where prion protein forms an equilibrium intermediate with enhanced beta-structure, potentially analogous to the conversion of the cellular form of the prion protein into the infectious form (PrP(C)-->PrP(Sc)). Furthermore, no direct interaction of either doppel protein with prion protein, either in the alpha-form or the beta-rich conformation, was detectable spectroscopically. These studies indicate that, in spite of the similarity in secondary structure between the doppel and prion protein, there are significant differences in their solution properties. The fact that neither doppel nor its mutant exhibited the alpha-->beta transformation of the prion protein suggests that this conversion property may be dependent on unique sequences specific to the prion protein.
Insights
Doppel protein, structurally similar to prion protein, is less stable and does not undergo the same alpha-to-beta transformation linked to neurodegenerative diseases. This suggests unique prion protein sequences are key to its disease-associated conformational changes.
Area of Science:
- Biochemistry
- Neuroscience
- Structural Biology
Background:
- Prion protein (PrP) and doppel (Dpl) are homologous proteins implicated in neurodegenerative diseases.
- The exact mechanisms underlying their roles in disease pathogenesis remain unclear.
- Understanding Dpl's biophysical properties can shed light on PrP's disease-associated functions.
Purpose of the Study:
- To investigate the conformational stability and unfolding mechanisms of murine doppel and a disulfide bond-deficient mutant.
- To compare the solution properties of doppel with those of prion protein under various conditions.
- To explore the potential for doppel to undergo the alpha-to-beta conformational transformation observed in prion protein.
Main Methods:
- Preparation of recombinant murine doppel and a mutant lacking one disulfide bond.
- Far-UV circular dichroism (CD) spectroscopy to monitor protein unfolding.
- Thermal and chemical denaturation studies using urea.
- Spectroscopic analysis to detect interactions between doppel and prion protein.
Main Results:
- Doppel and its mutant exhibit partial unfolding at elevated temperatures but fully unfold reversibly with urea.
- Doppel is significantly less stable than prion protein, with stability further reduced by disulfide bond removal.
- Both doppel forms unfold via a two-state mechanism, unlike prion protein which forms an intermediate.
- No direct spectroscopic interaction was detected between doppel and prion protein in different conformations.
Conclusions:
- Doppel possesses distinct solution properties compared to prion protein, despite secondary structure similarities.
- The absence of an alpha-to-beta transformation in doppel suggests this property is specific to prion protein.
- Unique sequences within prion protein may be critical for its disease-associated conformational conversion and neurotoxicity.