Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

Abstract

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore.

The structure of a far‐red fluorescent protein, AQ143, shows evidence in support of reported red‐shifting chromophore interactions

“…Many engineered far‐red FPs exhibit slow or incomplete maturation to the red chromophore, and it has recently been shown that maturation to the green and red chromophores in DsRed‐type FPs occurs via a branched pathway (i.e., the two forms of the chromophore are separate endpoints in chromophore maturation; the green is not an intermediate in the maturation to the red chromophore as had been previously proposed) . AQ143 is a DsRed‐type FP with a chromophore composed of a methionine/tyrosine/glycine triad that matures to both a green and a red chromophore (Fig.…”

“…mPlum, the furthest red‐shifted monomeric FP, has a similar hydrogen bonding interaction between Glu16 and the chromophore N‐acylimine, but is lacking a co‐ordinated water molecule to provide the second hydrogen bond. The importance of hydrogen bonds to the N‐acylimine was shown in mPlum variants, in which Glu16 is mutated to other residues including proline and glutamine, causing significant hypsochromic shifts to λ em …”

“…The importance of hydrogen bonds to the N-acylimine was shown in mPlum variants, in which Glu16 is mutated to other residues including proline and glutamine, causing significant hypsochromic shifts to k em . 18,25,26 Additionally, flexibility in the hydrogen bonding network to the phenolate oxygen of the chromophore, particularly via water-mediated hydrogen bonds, has been proposed to be responsible for extended stokes' shifts and significant bathochromic shifts to fluorescence emission. 8 The modeled cis chromophore, which we believe to be the fluorescent moiety, can accommodate two hydrogen bonds to the phenolate oxygen from the hydroxyl of Ser143 and a structural water molecule (Fig.…”

Monomerization of Far-Red Fluorescent Proteins

“…We reasoned that further stabilization of the cis chromophore would increase brightness, and so designed a first core library (cLibA) to target hotspot A, mutating trans -stabilizing amino acids, placing bulkier side chains into the trans pocket, and allowing varied hydrogen bonding geometries to the cis chromophore. A second core library (cLibB) targeted hotspot B along with two chromophore-backing positions (Gly28 and Met41 are implicated in maturation and color) (21, 23, 24). Two key features of this hotspot are a channel populated by structural water molecules that stretches to the protein surface, and Arg67, a key catalytic residue.…”

“…These deformations of the β-barrel are bisected by the attachment site of the C-terminal tail, and appear to be stabilized by intermolecular interactions between monomers across the AC interface. A break of the AC interface may destabilize the water channel, putting the chromophore environment into contact with bulk solvent, which would in turn interfere with chromophore maturation and quench fluorescence (23, 30). Indeed, mGinger1 and mKelly1 have eleven and six mutations respectively to residues that are in close proximity (4 Å) to structural waters in this channel and that are not a part of the AC interface (Figure S5).…”