Sarell, CJ, Karamanos, TK, White, SJ et al. (6 more authors) (2014) Distinguishing closely related amyloid precursors using an RNA aptamer. The Journal of Biological Chemistry, 289 (39). 26859 - 26871. ISSN 0021-9258
Abstract
Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β2-microglobulin (hβ2m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ2m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ2m with an EC50 of ∼200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-hβ2m complex, revealing that the aptamer binds to the face of hβ2m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ2m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | (c) 2014, Sarell, CJ, Karamanos, TK, White, SJ, Bunka, DH, Kalverda, AP, Thompson, GS, Barker, AM, Stockley, PG and Radford, SE. This is an Open Access article distributed in accordance with the Creative Commons Attribution (CC BY 3.0) licence, which permits others to distribute, remix, adapt, build upon this work, and license their derivative works on different terms, provided the original work is properly cited. |
Keywords: | Amyloid; amyloid Fibril; amyloid Precursor; aptamer; co-polymerization; protein aggregation; protein folding; RNA aptamer; structural biology; β2-Microglobulin; aptamers, nucleotide; humans; nuclear magnetic resonance, biomolecular; protein multimerization; beta 2-Microglobulin |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 19 Feb 2015 17:23 |
Last Modified: | 16 Nov 2016 09:54 |
Published Version: | http://dx.doi.org/10.1074/jbc.M114.595066 |
Status: | Published |
Publisher: | American Society for Biochemistry and Molecular Biology |
Identification Number: | 10.1074/jbc.M114.595066 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:83248 |