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Monomerization of far-red fluorescent proteins
Timothy M. Wannier
a,1,2
, Sarah K. Gillespie
a
, Nicholas Hutchins
a
, R. Scott McIsaac
b
, Sheng-Yi Wu
c
, Yi Shen
c
,
Robert E. Campbell
c,d
, Kevin S. Brown
e,f,g,3
, and Stephen L. Mayo
a,1
a
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;
b
Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, CA 91125;
c
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada;
d
Department of
Chemistry, The University of Tokyo, 113-0033 Tokyo, Japan;
e
Department of Chemical and Biomedical Engineering, University of Connecticut, Storrs, CT
06269;
f
Department of Physics, University of Connecticut, Storrs, CT 06269; and
g
Department of Marine Sciences, University of Connecticut, Groton, CT
06340
Contributed by Stephen L. Mayo, October 12, 2018 (sent for review June 13, 2018; reviewed by Amy E. Palmer and Vladislav V. Verkhusha)
Anthozoa
-class red fluorescent proteins (RFPs) are frequently used
as biological markers, with far-red (
λ
em
600
700 nm) emitting
variants sought for whole-animal imaging because biological tis-
sues are more permeable to light in this range. A barrier to the use
of naturally occurring RFP variants as molecular markers is that all
are tetrameric, which is not ideal for cell biological applications.
Efforts to engineer monomeric RFPs have typically produced dim-
mer and blue-shifted variants because the chromophore is sensi-
tive to small structural perturbations. In fact, despite much effort,
only four native RFPs have been successfully monomerized, leav-
ing the majority of RFP biodiversity untapped in biomarker devel-
opment. Here we report the generation of monomeric variants of
HcRed and mCardinal, both far-red dimers, and describe a compre-
hensive methodology for the monomerization of red-shifted olig-
omeric RFPs. Among the resultant variants is mKelly1 (emission
maximum,
λ
em
=
656 nm), which, along with the recently reported
mGarnet2 [Matela G, et al. (2017)
Chem Commun (Camb)
53:979
982], forms a class of bright, monomeric, far-red FPs.
fluorescent protein
|
red fluorescent protein
|
protein engineering
|
computational protein design
|
RFP
T
he development of red fluorescent proteins (RFPs) as tags
for molecular imaging has long focused on monomerization,
increased brightness, and pushing excitation and emission to
ever-longer wavelengths. These traits are desirable for live ani-
mal imaging because far-red and near-infrared light penetrates
tissue with minimal absorption in what is known as the near in-
frared window (
625
1,300 nm) (1, 2). Monomericity is impor-
tant because oligomerization of a fluorescent protein (FP) tag
can artificially aggregate its linked protein target, altering dif-
fusion rates and interfering with target transport, trafficking, and
activity (3, 4). Recently, a new class of infrared fluorescent
proteins (iRFPs) was developed from the bacterial phytochrome
(5, 6), but these require the covalent linkage of a small molecule
chromophore, biliverdin, limiting their use to cells and organisms
that make this molecule in sufficient quantity.
Anthozoa
-class
RFPs (such as mCherry and mKate) have the advantage that
the chromophore is created via a self-catalyzed reaction, neces-
sitating only molecular O
2
for chromophore formation (7), and
have been engineered to exhibit peak fluorescence at wave-
lengths as long as 675
685 nm (8, 9).
To our knowledge,
50 native RFPs and
40 chromoproteins
(CPs) with peak absorbance in the red or far-red (absorbance
maximum,
λ
abs
>
550 nm) have been described to date, but most
have not been extensively characterized because they are as a
class tetrameric and thus are less useful as biological markers
(10, 11). An underlying biological reason for the obligate tetra-
merization of native RFPs has been suggested but is not well
understood (12
15). Oligomerization does seem to play an im-
portant structural role, however, because breaking tetrameriza-
tion without abrogating fluorescence has proved difficult, and
successful monomerization has always led to either a hyp-
sochromic shift in
λ
em
or a decrease in brightness (16
19). Pre-
vious efforts to monomerize native RFP tetramers have relied on
lengthy engineering trajectories, with only four native RFPs having
been successfully monomerized before this work (Table 1). Gen-
erally, mutations are first introduced into protein/protein inter-
faces to weaken oligomerization, an inefficient process that
compromises fluorescence, and then random mutagenesis and
screening are used to isolate variants with partially recovered
fluorescence. After many such cycles, monomeric variants have
been found, but protein core and chromophore-proximal muta-
tions are invariably introduced, making it difficult to exert control
over the fluorescent properties of the resultant monomer. It is
thus difficult to know whether the poor spectroscopic character-
istics of engineered monomers are an unavoidable consequence of
monomerization or only the manifestation of a suboptimal evolu-
tionary path. The engineering of mScarlet, a bright red monomer
that was designed synthetically from previous RFP monomers, lends
evidence in support of the poor characteristics of monomers not
being intrinsic to the monomeric scaffold (20).
Here we present a comprehens
ive engineering strategy for
the monomerization of RFPs that differentiates itself by
treating separately the problems of protein stabilization, core
optimization, and surface design. We sample mutational space
Significance
All known naturally occurring red fluorescent proteins (RFPs), a
class that is desirable for biological imaging, are tetrameric,
limiting their usefulness as molecular fusion tags in in vivo
model systems. Here we explore protein variant libraries tar-
geted at monomerizing far-red RFP variants and describe a
generalizable method to monomerize RFPs of interest. This
method preserves the fluorescence of the molecule throughout
its monomerization, in contrast to break
fix methods, allowing
selective enrichment of bright, far-red monomeric variants.
Furthermore, we report four bright monomeric RFPs here,
which are among the most red-shifted of any monomeric
Aequorea victoria
-class FPs.
Author contributions: T.M.W., R.S.M., R.E.C., K.S.B., and S.L.M. designed research; T.M.W.,
S.K.G., N.H., S.-Y.W., and Y.S. performed research; T.M.W., Y.S., and K.S.B. analyzed data;
and T.M.W. and S.L.M. wrote the paper.
Reviewers: A.E.P., University of Colorado; and V.V.V., Albert Einstein College of Medicine.
The authors declare no conflict of interest.
Published under the
PNAS license
.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank,
www.wwpdb.org
(PDB ID code:
6DEJ
). GenBank IDs [accession nos.
MK040729
(mGinger),
MK040730
(mGinger2),
MK040731
(mKelly1), and
MK040732
(mKelly2)].
1
To whom correspondence may be addressed. Email: timothy_wannier@hms.harvard.edu
or steve@mayo.caltech.edu.
2
Present address: Department of Genetics, Harvard Medical School, Boston, MA 02115.
3
Present addresses: Department of Pharmaceutical Sciences, Oregon State University,
Corvallis, OR 97331; and School of Chemical, Biological, and Environmental Engineering,
Oregon State University, Corvallis, OR 97331.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1807449115/-/DCSupplemental
.
Published online November 13, 2018.
E11294
E11301
|
PNAS
|
vol. 115
|
no. 48
www.pnas.org/cgi/doi/10.1073/pnas.1807449115