Abstract
The formation of protein complexes is central to biology, with oligomeric proteins more prevalent than monomers. The coupling of functionally and even structurally distinct protein units can lead to new functional properties not accessible by monomeric proteins alone. While such complexes are driven by evolutionally needs in biology, the ability to link normally functionally and structurally disparate proteins can lead to new emergent properties for use in synthetic biology and the nanosciences. Here we demonstrate how two disparate proteins, the haem binding helical bundle protein cytochrome b 562 and the β-barrel green fluorescent protein can be combined to form a heterodimer linked together by an unnatural triazole linkage. The complex was designed using computational docking approaches to predict compatible interfaces between the two proteins. Models of the complexes where then used to engineer residue coupling sites in each protein to link them together. Genetic code expansion was used to incorporate azide chemistry in cytochrome b 562 and alkyne chemistry in GFP so that a permanent triazole covalent linkage can be made between the two proteins. Two linkage sites with respect to GFP were sampled. Spectral analysis of the new heterodimer revealed that haem binding and fluorescent protein chromophore properties were retained. Functional coupling was confirmed through changes in GFP absorbance and fluorescence, with linkage site determining the extent of communication between the two proteins. We have thus shown here that is possible to design and build heterodimeric proteins that couple structurally and functionally disparate proteins to form a new complex with new functional properties.
Highlights
Protein oligomerisation, commonly referred to as protein quaternary structure, is the association of specific individual polypeptide chains through defined intermolecular interactions to form a single multimeric complex (Goodsell and Olson, 2000; Nooren and Thornton, 2003; Ali and Imperiali, 2005)
We have previously proposed that such a positive synergistic effect is due to reduced water dynamics in channels leading to the superfolder version of green fluorescent protein (sfGFP) chromophore when homo-dimerisation occurs via residue 204 (Pope et al, 2020); the same may be occurring here
While traditionally protein engineering has focused on converting oligomeric proteins into monomers, especially with
Summary
Commonly referred to as protein quaternary structure, is the association of specific individual polypeptide chains through defined intermolecular interactions to form a single multimeric complex (Goodsell and Olson, 2000; Nooren and Thornton, 2003; Ali and Imperiali, 2005). Oligomerisation leads to functional features not available in monomers; they locally concentrate multiple active sites resulting in improved activity and enabling functional cooperativity whereby synergy [communication] between each polypeptide unit can positively or negatively regulate activity or even lead to new properties (Goodsell and Olson, 2000; Gwyther et al, 2019). While dimers represent the simplest protein oligomeric unit, they are the most frequently observed structural form in nature, with homo-dimers (comprising of the same polypeptide) dominating over hetero-dimer (composed of two different polypeptides) (Goodsell and Olson, 2000; Marianayagam et al, 2004; Mei et al, 2005)
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