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From single amino acid deletions to whole domain insertions; Engineering GFP through polypeptide backbone mutations

Arpino, James 2011. From single amino acid deletions to whole domain insertions; Engineering GFP through polypeptide backbone mutations. PhD Thesis, Cardiff University.
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With an ever-expanding protein engineering toolbox different mutational techniques can be used to engineer new or altered function into protein scaffolds without the restriction of sampling just simple substitution mutagenesis. These include approaches that target backbone as well as side chain changes, such as single amino acid deletions or whole domain insertion. The problem with utilizing these mutational approaches is the difficulty in predicting both local and global structural changes on changing the backbone conformation. The aim of this thesis is to demonstrate the tolerance and beneficial influence of backbone targeted mutations using a mutant library-based screening approach, and to provide an understanding of their mechanism of action. A directed evolution transposon-based approach was used to generate libraries of single amino acid deletion and whole domain insertions into enhanced green fluorescent protein (EGFP). The later involved the insertion of cyt b562 as the donating insert domain. Library analysis revealed a wide range of sites were sampled across the backbone of EGFP. Library screening revealed widespread tolerance of EGFP to single amino acid deletions. Using the crystal structure of EGFP determined here, it was found that that loop regions where particularly tolerant. Two variants with residues G4 or A227 deleted conferred increased protein fluorescence to cell cultures with respect to EGFP. Spectral characterization and unfolding experiments identified that rather than altering the fluorescent properties of EGFP the mutations elicited their effects through altered protein folding and stability. Screening of the domain insert library revealed that sites spread along the backbone of EGFP were tolerant to cyt b562 insertion. Particularly tolerant were loops and the C-terminal end of β-strand 7, with the linker sequences playing a key role. One integral domain fusion scaffold, termed CG6, was identified in which the functions of the two individual domains were highly coupled. CG6 exhibited almost 100% fluorescence quenching upon the binding of haem to the cyt b562 domain. CG6 was also shown to potentially act as a sensor for redox state and reactive oxygen species such as H2O2 via a haem-dissociation dependent mechanism. The structure of CG6, determined by X-ray crystallography, provided the molecular basis for the functional coupling of the two domains. Critical was the side-by-side domain arrangement caused by differential linker lengths at the pivot position re-enforced by a domain-domain interface that placed the chromophores within 17-18Å of each other. Further rational design of a cyt b562-EGFP integral fusion scaffold (CG15) was performed to create novel ratiometric fluorescent redox sensors, termed CG15CC variants. The CG15CC variants have been shown to have the most reducing redox midpoint potentials of any protein based redox sensor studied to date. One of the CG15CC variants also has the fastest redox kinetics observed to date. The survey of the tolerance and influence of single amino acid deletions in EGFP conducted here has highlighted the potential beneficial nature of deletion mutagenesis and has helped provide a molecular understanding of their effect. Through domain insertion mutagenesis and retrospective structure analysis the mechanism behind the functional coupling of two domains has been described and will also help guide future work in the development of novel biomolecular switches.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Biosciences
Uncontrolled Keywords: EGFP; Integral domain fusion architecture; Directed Evolution; Transposon
Date of First Compliant Deposit: 30 March 2016
Last Modified: 19 Mar 2016 22:23

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