Abstract |
The N-terminal 'unstructured' region of the human prion protein [PrP((90-231))] is believed to play a role in its aggregation because mutations in this region are associated with seeding-independent deposition disorders like Gerstmann-Straussler-Scheinker disease (GSS). One way of examining the effects of such mutations is to search combinatorially derived libraries for sequence variants showing a propensity to aggregate and/or the ability to interact with prion molecules folded into a beta-sheet-based conformation (i.e., beta-PrP or PrP(Sc)). We created a library of 1.8x10(7) variants randomized between positions 101 and 112, displayed it on filamentous bacteriophage, and 'spiked' it with a approximately 25% population of phages-bearing wild-type prion (wt-PrP). Screening was performed through four rounds of biopanning and amplification against immobilized beta-PrP, and yielded three beta-PrP-binding populations: wt-PrP (26% representation) and two non-wt-PrP variants ( approximately 10% and approximately 64% representation, respectively). The remarkable enrichment of one non-wt-PrP variant (MutPrP) incorporating residues KPSKPKTNMKHM in place of KGVLTWFSPLWQ, despite its initial representation at a 5 million-fold lower level than wt-PrP, caused us to produce it and discover: (i) that it readily aggregates into thioflavin-T-binding amyloids between pH 6.0 and 9.0, (ii) that it adopts a soluble beta-sheet based monomeric structure at pH 10.0, (iii) that it is less thermally stable and more compact than wt-PrP, and (iv) that it displays significantly greater resistance to proteolysis than wt-PrP. Our results suggest that sequence variations in the 101-112 region can indeed predispose the prion for aggregation.
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Authors | Archana Verma, Swati Sharma, Nirmal Kumar Ganguly, Siddharta Majumdar, Purnananda Guptasarma, Manni Luthra-Guptasarma |
Journal | The international journal of biochemistry & cell biology
(Int J Biochem Cell Biol)
Vol. 40
Issue 4
Pg. 663-76
( 2008)
ISSN: 1357-2725 [Print] Netherlands |
PMID | 18023239
(Publication Type: Journal Article)
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Chemical References |
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Topics |
- Amino Acid Sequence
- Amyloid
(metabolism, ultrastructure)
- Chromatography, Gel
- Circular Dichroism
- Electrophoresis, Polyacrylamide Gel
- Humans
- Hydrogen-Ion Concentration
- Microscopy, Electron, Transmission
- Molecular Sequence Data
- Prions
(chemistry, genetics, metabolism, ultrastructure)
- Protein Folding
- Sequence Analysis, Protein
- Sequence Homology, Amino Acid
- Solubility
- Spectrometry, Fluorescence
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Spectroscopy, Fourier Transform Infrared
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