Instability of familial spongiform encephalopathy-related prion mutants

Abstract

We examined the influence of D177N (D178N in humans) mutation on the conformational stability of the S2 region of moPrP C with varying pHs by using the SDSL-ESR technique. The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT*. The ESR spectrum of D177N did not change when pH in the solution decreased to pH 4.0. Our results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. Moreover, the line shape of the ESR spectrum obtained from H176S neighboring the salt bridge linked to the S2 region was similar to D177N. These results indicate that the protonation of H176 is strongly associated with the stability of S2 region. These findings are important for understanding the mechanism by which the disruption of the salt bridge in the S2 region forms the pathogenic PrP Sc structure in hereditary prion disease.

Molecular Dynamics as an Approach to Study Prion Protein Misfolding and the Effect of Pathogenic Mutations

“…The interactions with both strands of the native β-sheet may be disrupted by the D178N mutation, thereby potentially affecting its stability. Spin-labeling ESR studies of D178N PrP indeed showed that the D178N mutation increases instability in S2 [139]. Crystal structures of D178N PrP with either M129 or V129 show little difference with the overall static fold of WT PrP [75].…”

A novel copper(II) coordination at His186 in full-length murine prion protein

“…cDNA encoding mouse PrP (residues 23-231) was cloned into BamHI/EcoRI sites of pRSETb as described previously [19][20][21][22]27]. In the plasmid encoding moPrP, four amino acid residues, D143, Y148, E151 in the H1 region, Y156 in the loop between H1 and S2 and T189 in the H2 region, were changed to cysteine residues for the labeling site as indicated in Fig.…”

“…In the plasmid encoding moPrP, four amino acid residues, D143, Y148, E151 in the H1 region, Y156 in the loop between H1 and S2 and T189 in the H2 region, were changed to cysteine residues for the labeling site as indicated in Fig. 1A using the PCR-based site-directed mutagenesis method described previously [19][20][21][22].…”

“…The expression and purification of recombinant moPrP mutants were carried out as described previously [21][22][23][24]. To label the moPrP C mutants with …”

“…Recently, to obtain information about the pH-induced conformational changes, we employed cysteine-scanning site-directed spin labeling (SDSL) combined with electron spin resonance spectroscopy (ESR), and analyzed the pH-induced mobility changes in one -helix (Helix1) and two -sheets (Sheet1 and Sheet2) of mouse PrP C (moPrP C ) [19][20][21][22]. Our experimental data using cysteine-scanning spin labeling for 19 cysteine mutants of recombinant moPrP C clearly demonstrated the presence of three pH-sensitive sites in moPrP C .…”