Identification and characterization of a spontaneously aggregating amyloid-forming variant of human PrP((90-231)) through phage-display screening of variants randomized between residues 101 and 112.

Verma, Archana and Sharma, Swati and Ganguly, N K and Majumdar, Siddharta and Guptasarma, Purnananda and Luthra-Guptasarma, Manni (2008) Identification and characterization of a spontaneously aggregating amyloid-forming variant of human PrP((90-231)) through phage-display screening of variants randomized between residues 101 and 112. The international journal of biochemistry & cell biology, 40 (4). pp. 663-76. ISSN 1357-2725

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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.

Item Type:	Article
Additional Information:	Copyright of this article belongs to Elsevier Science.
Uncontrolled Keywords:	Prion aggregation; Protein misfolding; Phage display; Sequence variants; Amyloid formation; Conformational transition
Subjects:	Q Science > QD Chemistry
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	21 Feb 2012 18:06
Last Modified:	02 Apr 2012 07:17
URI:	http://crdd.osdd.net/open/id/eprint/1076

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