Slow spontaneous α-to-β structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

Abstract

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.

N-terminal Domain of Prion Protein Directs Its Oligomeric Association

“…Although we do not observe a specific cellular toxicity of this β-PrP 23–231 preparation, several recombinant β-oligomers are reported to be toxic to neuronal cell cultures ( 13 , 29 , 30 , 66 , 74 , 75 ). The variety of cell types tested, the various preparations of recombinant PrP oligomers, and the considerable experimental variation may explain variability in toxicity, but it is also probable that subtle differences between the various species under investigation can result in large differences in biological effect.…”

“…The complementary solution methods used here have proved necessary to enable the protein association state to be verified independently of the spectroscopic signal. The volume and size measurements indicated by these different techniques are similar to a number of other discrete non-fibrillar oligomers formed from recombinant PrP under various in vitro folding conditions ( 13 , 21 , 23 – 27 , 29 – 32 , 66 ).…”

Location of the cross‐β structure in prion fibrils: A search by seeding and electron spin resonance spectroscopy

“…Another physical method, spin-label electron spin resonance (ESR) spectroscopy, has also been used as a spectroscopic ruler to locate the cross-β structure in inhomogeneous fibrils or oligomers and to explore the structural conversion and aggregation of proteins or peptides such as amyloid β-peptides, tau, transthyretin, and prion protein. [19][20][21][22][23][24][25][26][27][28] In this method, nitroxide spin-labels are introduced into the prion molecule, and one can measure the spin-spin interaction between two unpaired electrons at a distance within 20 Å using continuouswave ESR (CW-ESR) and 15 to 60 Å using double electron-electron resonance (DEER) ESR. 13,29,30 To investigate biomolecules with spin-labeled ESR, a nitroxide-based probe is attached through site-directed spin-labeling (SDSL) mutagenesis.…”

Directly monitor protein rearrangement on a nanosecond-to-millisecond time-scale

“…For example, a disulfide bond stabilizes prion protein. Without the disulfide bond, the second α-helix of prion protein is unfolded at room temperature, neutral pH, and in the absence of denaturant, and this partially unfolded prion protein gradually assembles into β-oligomers or fibrils 4 , 5 . The misfolding kinetics is driven by hydrophobic interaction and can be tuned by salt concentration in the protein solution.…”