We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1.
Our basic strategy for decreasing the oligomeric state of DsRed was to replace key dimer interface residues with arginine. When the targeted residue interacts with the identical residue of the dimer partner through symmetry, the high energetic cost of placing two positive charges in close proximity should disrupt the interaction. Such a strategy disrupted the weak tendency of wtGFP to dimerize, without any deleterious effects on GFP maturation or brightness (Zacharias et al. 2002).
[Dimer1 is an intermediate DsRed mutant in the development dimer2 and mRFP1]. Dimer1 was somewhat better than wild-type DsRed both in terms of brightness and rate of maturation but had a substantial green peak equivalent to that of DsRed.T1. Dimer1 was also somewhat blue-shifted with an excitation maximum at 551 nm and an emission maximum at 579 nm.
Based on our early results, it seemed as though engineering a true monomer of DsRed might be impossible and therefore we pursued an alternate approach. The basic strategy was to fuse two copies of our best AC dimer with a polypeptide linker such that the critical dimer interactions could be satisfied through intramolecular contacts with the tandem partner encoded within the same polypeptide. With our optimized dimer2 we constructed a series of tandem constructs with linkers of varying lengths (9, 12, 13, or 22 aa) and a sequence similar to a known protease-resistant linker. Of these four, only the tandem construct with the nine-residue linker was notable for its somewhat slower maturation. The other three constructs were practically indistinguishable, and the tandem construct with the 12-residue linker, designated tdimer2(12), is currently our preferred construct because it has the shortest linker.