Structure–Function Relationships in Fluorescent Marker Proteins of the Green Fluorescent Protein Family - Fluorescent Proteins: From Understanding to Design - page 241

including tissue imaging, monitoring of gene activity, and highlighting of cells and
cellular compartments. However, oligomerization can be detrimental in studies
involving FP fusions with other proteins because it may interfere with the function
of the fusion partner [ 46 ]. Once expressed, formation of dimers or higher-order
oligomers induced by the FP part of the fusion construct may produce atypical
localization, or alter normal function. The basic strategy for overcoming oligomer-
ization is to modify the FP amino acid sequence to include residues that disrupt the
binding interfaces between the protomers, a procedure that may greatly vary in
complexity depending on the nature and origin of the FP. For GFP-based variants,
dimerization can be either significantly reduced or even completely eliminated by
replacing hydrophobic amino acid side chains in the dimer interface with positively
charged residues at several key sequence positions. The three most successful
mutations, in increasing order of effectiveness, are Phe223Arg, Leu221Lys, and
Ala206Lys, where the nonpolar amino acids phenylalanine, leucine, and alanine are
replaced by hydrophilic alternatives (arginine or lysine) [ 50 ]. In GFP, a single
mutation (Ala206Lys) was sufficient for monomerization [ 51 ].
Creating FP monomers from tetrameric reef coral and sea anemone proteins has
proven far more difficult. Only in a few favorable cases, tetrameric anthozoan FPs
have been turned into fairly bright monomers by single amino acid exchanges in
each of the two interfaces between -barrels [ 52 ]. In less favorable cases, however,

disruption of the tetramer resulted in drastic, sometimes even complete loss of
fluorescence [ 53 , 54 ]. To recover the emission, the dysfunctional protein was
subsequently subjected to multiple turns of random mutagenesis followed by
screening for brighter clones.
The red FP (RFP) DsRed, for example, is a tightly associated tetramer even at
exceedingly low concentrations, and cannot be dissociated without irreversible
denaturation of the individual polypeptides [ 33 ]. To remove the interactions of
each DsRed protomer with its two neighbors, one through a hydrophobic interface
and the other through a hydrophilic interface, 33 mutations were required [ 54 ].
Other Anthozoa proteins have weaker subunit interactions that, at first sight,
may prove easier to break apart into functional monomers. For eqFP611, an RFP
from the sea anemone Entacmaea quadricolor , functional dimers, denoted by
d1eqFP611 and d2eqFP611 were simply obtained by the single mutations
Val124Thr or Thr122Arg in the A/B interface, respectively [ 53 ]. However, a
functional monomer was only obtained after a total of seven rounds of random
mutagenesis and four rounds of multiple site-directed mutagenesis. Compared with
wild-type eqFP611, the bright red-fluorescent monomer, denoted as mRuby, con-
tains altogether 28 amino acid replacements and is shorter by four amino acids [ 55 ].
Another technique for generating “pseudo” monomers from dimeric FPs
involves linking two copies of the FP cDNA with a short intervening DNA
sequence encoding simple neutral or hydrophilic amino acids (glycine, alanine,
and serine) to form so-called tandem dimers. Upon expression in live cells, the
fused FPs preferentially interact with each other to form intramolecular dimeric
units that perform essentially like monomers, although they have twice the molec-
ular mass. This method was successfully applied with HcRed by fusing two copies

b