Related Experiment Videos
NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231)
1Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
Abstract:
The recombinant murine prion protein, mPrP(23-231), was expressed in E. coli with uniform 15N-labeling. NMR experiments showed that the previously determined globular three-dimensional structure of the C-terminal domain mPrP(121-231) is preserved in the intact protein, and that the N-terminal polypeptide segment 23-120 is flexibly disordered. This structural information is based on nearly complete sequence-specific assignments for the backbone amide nitrogens, amide protons and alpha-protols of the polypeptide segment of residues 121-231 in mPrP(23-231). Coincidence of corresponding sequential and medium-range nuclear Overhauser effects (NOE) showed that the helical secondary structures previously identified in mPrP(121-231) are also present in mPrP(23-231), and near-identity of corresponding amide nitrogen and amide proton chemical shifts indicates that the three-dimensional fold of mPrP(121-231) is also preserved in the intact protein. The linewidths in heteronuclear 1H-15N correlation spectra and 15N[1H]-NOEs showed that the well structured residues 126-230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23-231) outside of this domain.
Insights
Recombinant murine prion protein (mPrP) structure was analyzed using NMR. The study found the C-terminal domain retains its globular structure, while the N-terminal region remains disordered in intact mPrP.
Area of Science:
- Biochemistry
- Structural Biology
- Neuroscience
Background:
- Prion diseases are linked to misfolded prion proteins.
- Understanding prion protein structure is crucial for disease mechanism elucidation.
- The murine prion protein (mPrP) serves as a model system.
Purpose of the Study:
- To determine the structural integrity of the full-length recombinant murine prion protein (mPrP(23-231)).
- To investigate the structural state of the N-terminal flexible region (residues 23-120) in the context of the intact protein.
- To confirm the preservation of the globular C-terminal domain structure (mPrP(121-231)) within the full-length protein.
Main Methods:
- Expression of uniformly 15N-labeled recombinant murine prion protein (mPrP(23-231)) in E. coli.
- Nuclear Magnetic Resonance (NMR) spectroscopy, including heteronuclear 1H-15N correlation spectra and 15N[1H]-NOEs.
- Sequence-specific assignments for backbone amide nitrogens, amide protons, and alpha-protons of the C-terminal domain.
Main Results:
- NMR experiments confirmed that the globular three-dimensional structure of the C-terminal domain (mPrP(121-231)) is preserved in the intact mPrP(23-231).
- The N-terminal polypeptide segment (residues 23-120) was found to be flexibly disordered.
- Analysis of linewidths and correlation times indicated that residues 126-230 are well-structured with typical globular protein dynamics, while residues outside this domain exhibit faster dynamics.
Conclusions:
- The intact recombinant murine prion protein (mPrP(23-231)) maintains the structured C-terminal domain observed in isolated fragments.
- The N-terminal region of mPrP(23-231) is intrinsically disordered, suggesting a flexible role in protein function or interaction.
- These findings provide critical structural insights into the full-length prion protein, relevant for understanding its biological role and potential involvement in prion diseases.