Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629288
Title: PNA-protein conjugates for nano-scale modeling of protein aggregates
Author: Gholami, Z.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2013
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Abstract:
Programmable assembly of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this research, the unique characteristics of PNA were applied to create controllable protein assemblies directed by precise PNA-DNA hybridization. The signatures of assembly were studied via FRET, providing a powerful tool which should be effective in live system imaging. Two model systems were developed in this study. In the first model system, site-selective conjugation of monomeric teal fluorescent protein (mTFP) to PNA was achieved by covalent linkage of mTFP to PNA via expressed protein ligation. The mTFP-PNA conjugates were efficiently aligned on a DNA beacon, to create a hetero-FRET system. The FRET indicated by decrease of fluorescence intensity and lifetime of the donor and an increase of donor anisotropy. The assembly of similar multiple mTFP-PNA constructs on DNA scaffolds provided dimeric and oligomeric forms which were studied by SEC-HPLC and SDS-PAGE. A decrease of anisotropy was exhibited due to homo-FRET following induced formation of dimers and oligomers. In the second model system, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers and oligomers. The site-selective covalent linkage of peptide nucleic acid (PNA) to SNAP protein was achieved by modification of PNA with O6-benzyl guanine (BG) which is a specific substrate for SNAP. The modified BG-PNA has been labeled with Atto dyes and thereafter, chemo-selectively conjugated to SNAP protein. Efficient assembly into dimeric and oligomeric forms were observed using SEC-HPLC and SDS-PAGE. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. These systems could readily extend from homodimers and oligomers to binary, ternary, and higher oligomer systems containing any number of different dyes or fluorescent proteins in precisely engineered arrangements.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.629288  DOI: Not available
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