821
Structure and dynamics of green fluorescent protein
George N Phillips Jr
Many marine organisms are luminescent. The proteins that
produce the light include a primary light producer (aequorin or
luciferase) and often a secondary photoprotein that red shifts
the light for better penetration in the ocean. Green fluorescent
protein is one such secondary protein. It is remarkable in that
it autocatalyzes the formation of its own fluorophore and thus
can be expressed in a variety of organisms in its fluorescent
form. The recent determination of its 3D structure and
other physical characterizations are revealing its molecular
mechanism of action.
Addresses
Department of Biochemistry and Cell Biology, Rice University,
Houston, TX ?7005-1899, USA; e-mail: georgep@rice.edu
Current Opinionin StructuralBiology 1997, 7:821-82"7
http://biomednet.com/elecref/0959440XO0700821
© Current Biology Ltd ISSN 0959-440X
Abbreviation
GFP green fluorescent protein
Introduction
Bioluminescence was written about as early as the first
century AD by Pliny the Elder, who noticed the bright
light eminating from some jellyfish [1]. There appear to
be many independent evolutionary origins of luminous
species [2], some of which have primary light-generating
reactions followed by color-shifting secondary proteins.
Green fluorescent protein (GFP) is a spontaneously
fluorescent protein isolated from marine organisms, such
as certain jellyfish and sea pansies. It converts the blue
chemiluminescence of the primary proteins, aequorin or
luciferase, into green fluorescent light [3,4], presumably
to reduce scattering and hence improve penetration of the
light over longer distances.
T h e molecular cloning of G F P c D N A from the Pacific
jellyfish Aequorea ~ictoHa [5] and the demonstration by
Chalfie et al. [6] that this G F P can be functionally
expressed in bacteria have opened exciting new avenues of
investigation in cell, developmental and molecular biology,
as pointed out in prior Teviews [7,8"]. A very dramatic
recent example is the production of transgenic green mice
[9]. Knowledge of the 3D structures of G F P is helping
to understand the basic photochemistry of this protein,
and the desire for specially engineered fluorescent proteins
has led at least half a dozen laboratories to carry out
structural studies on G F P and its mutants. This review will
highlight the relationship between the structure, function
and dynamics of GFP.
Spectral and physical properties of G F P
T h e wild-type fluorescence absorbance/excitation peak
is at 395nm, with a minor peak at 4 7 5 n m (extinction
coefficients o f - 30 000 and - 7,000 M -1 cm -1, respectively)
[10]. T h e normal emission peak is at 508 nm. Interestingly,
continued excitation leads to a decrease over time of the
3 9 5 n m excitation peak and a reciprocal increase in the
4 7 5 n m excitation peak [11]. This interconvcrsion effect
is especially evident with irradiation of G F P with UV
light. Recently, femtosecond time-resolved spectroscopic
studies have revealed that the two states corresponding to
the two major absorption bands can interconvert quickly
in the excited state, and the presence of an isotope
effect implicates proton m o v e m e n t in the interconversion
lie.,13.].
Because crystalline G F P exhibits a nearly identical
fluorescence spectrum and lifetime to that in aqueous
solution [14], and fluorescence is not an inherent property
of the isolated fluorophore, the elucidation of its 3D
structure helps provide an explanation for the generation
of fluorescence in the mature protein, as well as the
mechanism of autocatalytic fluorophore formation. Fur-
thermore, the development of fluorescent proteins with
varied emission and excitation or other characteristics
would dramatically expand their biological applications as
intracellular reporters.
T h e ~-can structure of G F P
T h e structure of the wild-type protein was originally
solved by Yang et al. [15 °°] and that of the Ser65--+Thr
mutant was solved by Ormo et al. [16"]. T h e monomer
structure consists of a quite regular 13 barrel with 11
strands on the outside of a cylinder (Figure 1). T h e
cylinder has a diameter o f - 3 0 ~ and a length of-40/i~.
Inside the cylinder sits the fluorescent center of the
molecule - - a modified tyrosine sidechain and cyclized
protein b a c k b o n e - - a s a part of an irregular {x-helical
segment. Small sections of c~-helix also form caps on
the ends of the cylinders. This motif, with a single
a-helix inside a very uniform cylinder of 13-sheet structure,
represents a new class of protein fold, which we have
named the ']3-can'.
T h e regularity of the 13-can of G F P is quite remarkable.
T h e eleven strands of the sheet form an almost seam-
less symmetrical structure, the only irregularities being
between two of the strands. In fact, the structure is so
regular that water molecules on the outside of the can
also form 'stripes' around the surface of the cylinder. T h e
tightly constructed 13 barrel would appear to serve the
role of protecting the fluorophore well, providing overall
822 Proteins

Figure 1

(~ (b)

(c)

Current Opinion in Structural Biology

The cylindrical ~-can structure of GFP [15°']. (a) End-on view. (b) Side view. Eleven strands of ~-sheet form an antiparalle113 barrel with short
(~ helices forming lids on each end. The fluorophore is inside the can, as a part of a distorted cc helix which runs along the axis of the cylinder.
(c) GFP usually forms dimers in the crystal, aligned largely along the sides of the cylinders. Figure generated using Ribbons, PDB entry code
1 GFL.

stability and resistance to unfolding caused by heat and T h e known proteins that most closely resemble the [3-can
denaturants [17]. fold of G F P are porin, which has not 11 but 16 antiparallel
 Structure and dynamics of green fluorescent protein Phillips 823

Figure 2

Gin94 O_(
NH~
t NH--
I
I
| .J"
,H2N--~ Arg96
NH 2

"•N
HN...~' N
o. .J -°'y
H ,~'N--Phe o_(
His148 H OH
i ~ ...-H2N

"~ I NH 2
, , ( ~ ~ ' J ~ N - - G l y

Neutral Form
H-O
OH I
HO ,'

Anionic Form
Current Opinion in Structural Biology

Schematic diagram of the resonant forms of the fluorophore with nearby basic amino acids, His148, Gin94, and Arg96, and the acid Glu222.
The bases appear to stabilize anionic oxygen atoms at the opposite ends of the fluorophore, and the acid forms a hydrogen bond with the
hydroxyl of Ser65. Interactions between fluorophore and amino acids are shown with dotted lines.

strands and has no 'lids' at the ends of the barrel [18], and
strepavidin, which is a smaller, eight-stranded antiparallel
[3 barrel [19]. Unlike strepavidin, however, both GFP and
porin have water molecules inside the barrel, as well as
small segments of polypeptide chain inside. In the case
of porin, the function of which is to allow passage of small
molecules through its center, the design needs to be open,
whereas the function of GFP is better served by a closed
structure. Because of the smaller number of strands in
streptavidin, and hence the smaller inside diameter, its
center consists simply of sidechains originating from the
staves of the barrel.
The fluorophore and its environment
Analysis of a hexapeptide derived by proteolysis of
purified GFP has led to the prediction that the flu-
orophore originates from an internal Ser-Tyr-Gly se-
quence which is post-translationally modified to a 4-
(p-hydroxybenzylidene)-imidazolidin-5-one structure [20].
Studies of recombinant GFP expression in E. coil have
led to a proposed sequential mechanism initiated by a
rapid cyclization between Set65 and Gly67 to form a
imidazolin-5-one intermediate, followed by a much slower
(hours) rate-limiting oxidation of the Tyr66 sidechain by
02 [21,22°]. Combinatorial mutagenesis suggests that the
Gly67 is required for formation of the fluorophore [23].
While no known cofactors or enzymatic components are
required for this apparently autocatalytic process, it is
rather thermosensitive, with the yield of fluorescently
active to total GFP protein decreasing at temperatures
>30°C [24]. Once produced, however, GFP is quite
thermostable.
824 Proteins
The critical fluorophore-forming sequence, Ser-Tyr-Gly,
occurs frequently in proteins. How is it that cyclization
occurs in GFP, but not in other proteins? It would appear
that two factors are required for fluorophore formation:
close proximity of the backbone atoms of residues 65 and
67; and acid/base chemistry to catalyze the cyclicization.
Close proximity is achieved by the removal of steric
hindrance from the sidechain at position 67, by using
a glycine residue. In fact, despite many mutations at
position 67, no functionally fluorescent GFPs have been
found with anything but glycine at position 67. Amongst
the proteins in the protein data bank with a Ser-Tyr-Gly
sequence, 10 out of 206 found have the required proximity
yet no fluorophore is formed [25]; therefore steric factors
seem to be necessary but not sufficient for cyclization.
Arginine at position 96 is close by and could act as a
base, withdrawing electrons by hydrogen bonding with
the carbonyl oxygen of Ser65 and activating the carbonyl
carbon for nucleophilic attack by the amide nitrogen of
Gly67. Aspects of this scheme have been supported by
ab initio calculations, and by database searches of similar
compounds and protein sequences [26].
As model compounds identical to the hydroxyphenyl
imidizolidinone core of the fluorophore have been synthe-
sized and shown N O T to be significantly fluorescent in
solution [27], the protein and its strategically placed acids
and bases at the edges of the fluorophore are implicated in
providing key resonance stabilization and immobilization.
The remarkable cylindrical fold of the protein seems
ideally suited for this function of the protein. Together
with the short a helices and loops on the ends, the barrel
structure forms a single compact domain and does not have
obvious clefts for easy access of diffusablc ligands to the
fluorophore.
The fluorophorc is protected from collisions with fluores-
cence quenchers such as molecular oxygen (Kbm<0.004
L/mol-s) [28] and hence reduction of the quantum yield
of green photons that are produced from the excited state.
The lack of quenching of the fluorophore by molecular
oxygen can be explained by one of two mechanisms.
Either the [3-can structure provides significant barriers
to penetration by oxygen, or the fluorophore is tightly
held by local interactions. The observation that the
fluorophore is near the geometrical center of the can
supports the notion that the can structure protects the
fluorophore from diffusional penetrative quenching and/or
protects the organism from eventual and unavoidable
free-radical reactions begun by photochemical processes
at the fluorophore by contained self-destruction. The
crystallographic temperature factors are indeed lowest in
the center of the can, implying more rigidity, but it is
often the case that centers have low mobility within
protein crystal structures, and therefore this cannot be
taken as proof of a special design. GFP from the sea
pansy (Renilla reniformis) may have even more specialized
structures for maintaining the rigidity of the fluorophore,
as its absorption and emission spectra are essentially mirror
images [ 4 ] - - a hallmark of highly immobilized fluorescent
molecules.
Most of the other polar residues in the pocket form
an extensive hydrogen-bonding network on the side of
Tyr66 that requires abstraction of protons in the oxidation
proccss. It is tempting to speculate that these residues,
particularly Arg96, help abstract the protons. As for the
mutants, atoms in the sidechains of Thr203, Glu222,
and I1e167 are in van der Waals contact with Tyr66,
therefore mutation of these would have direct steric
effects on the fluorophore and would also change its
electrostatic environment if the charge were changed, as
has been suggested previously [29]. It seems probable that
other mutations of the residues identified to be near the
fluorophore would also have an effect on the absorption
and/or emission spectra.
Structures of variant G F P s
The location of certain amino acid sidechains within
the vicinity of the fluorophore also begins to explain
the fuorescence and the behavior of certain mutants of
GFP. At least two resonant forms of the fluorophore can
be drawn, one with a partial negative charge on the
benzyl oxygen of Tyr66, and one with the charge on the
carbonyl oxygen of the imidizolidone ring. Interestingly,
basic residues appear to form hydrogen bonds with
each of these oxygen atoms, His148 with Tyr66 and
Gin94 and Arg96 with the imidizolidone. These basis
residues presumably act to stabilize and possibly further
delocalize the charge on the fluorophorc (Figure 2).
The most probable possibility for charge changes is the
Tyr66 distal oxygen-Hisl48 donor/acceptor pair, together
with the Glu222-Ser65 interactions [30°°]. Perturbation
of the interaction by mutation to histidine at position
66 results in only the high-energy absorption peak
and a blue-shifted emission band, whereas disruption
of the Set65 hydroxyl-Glu222 interactions result in a
red-shifted absorption peak and an unchanged emission
spectrum. The Tyr66--~Phe mutation has been reported to
have dramatically reduced fluorescence [21], presumably
due to its inability to form the anionic form of the
fluorophore. Analysis of other mutants appears to support
the relationship between charge on the fluorophore and
the absorption/excitation spectra [31].
The availability of E. coli clones expressing GFP has led
to extensive mutational analysis of GFP, with more crystal
structures now starting to appear that test ideas about
the designed rearrangements of sidechains resulting from
mutagenesis (Table 1). Screens of random and directed
point mutations for changes in fluorescent behavior
have uncovered a number of informative amino acid
substitutions. Mutation of Ser65 to threonine, alanine,
cysteine or leucine causes a loss of the 395 nm excitation
peak with a major increase in blue (475nm) excitation
[23,32]. When combined with Ser65 mutants, mutations
 Structure and dynamics of green fluorescent protein Phillips 825

at other sites near the fluorophore such as Va168--+Leu
and Ser72-->Ala can further enhance the intensity of green
fluorescence produced by excitation at 4 8 8 n m [23,33].
Amino acid substitutions significantly outside this region,
however, also affect the proteins spectral character. For
example, Ser202--+Phe and Thr203-+Ile both cause the
loss of excitation in the 475 nm region with preservation
of 395 nm excitation [6,21,29]. Ile167-+Thr results in a
reversed ratio of 395 to 4 7 5 n m sensitivity [11], whereas
Glu222--+Gly is associated with the elimination of only
the 395 nm excitation [29]. T h e pH dependence of the
excitation bands at 395 and 475 nm [17] is almost certainly
due to His148, the N atom of which is 3 . 3 ~ from the
Tyr66 hydroxyl oxygen atom of the fluorophore, although
N M R pK a measurements or mutagenesis studies would be
needed for confirmation.
Structure-based engineering of GFP
Mutations in regions of the sequence adjacent to the
fluorophorc, that is, in the range of positions 65-67, have
been systematically explored [23], some have significant
wavelength shifts and most suffer a loss of fluorescence
intensity. For example, mutation of the central tyrosine to
phenylanine or histidine shifts the excitation bands, but
an overall loss of intensity occurs. Secondary mutations to
compensate for the deleterious intensity effects may also
now be possible. T h e Ser65--+Thr mutant is particularly
interesting because of its reported increase in fluorescence
intensity [21,32]. On the one hand, the mechanism
for increased fluorescence may be reduced collisional
quenching, as the additional methyl group may make
for better packing in the interior of the protein. On the
other hand, the effect has been suggested to be due to
improved conversion of the tyrosine to dehydrotyrosinc.
This is unlikely, however, as essentially we see fully
cyclized structure in the wild-type protein. This effect
is most likely produced by increased expression and/or
folding of the protein. T h e report of improvements in
G F P by D N A shuffling [34], comprising the mutations
Phe99--->Ser, Met153--->Thr and Val163--+Ala, as numbered
in the TU#58 system [6], are difficult to explain simply
on the basis of the structure. Positions 153 and 163 are
on the surface of the protein and may exert their effects
via improved solubility and/or reduced aggregation. At
first glances, the Phe99--+Ser mutation would appear to
destabilize the core of the protein, and at present we have
no good ideas how it would improve the system.
GFP truncation and fusion constructs
Truncation of more than seven amino acids from the C
terminus or more than the N-terminal methionine leads
to total loss of fluorescence [35]. T h e s e N- and C-termini
truncation studies and the fluorescent fusion products are
now understandable, given the structure of the protein. As
the C terminus loops back outside the cylinder, and the
last seven or so amino acids are disordered, their presence
shouldn't be critical, and further addition would seem to
be easily tolerated. T h e s e residues do not form a stave of
the barrel. T h e role of the N terminus is a little less clear,
as the first strand in the barrel does not begin until amino
acid 10 or 11. Thus, barrel formation does not require the
N-terminal region. T h e N-terminal segment is, however,
an integral part of the cap on one end of the protein, and
may be essential in folding events or in protecting the
fluorophore. Again, extensions at the N terminus would
not disrupt the motif structure of the protein. In fact,
the G F P crystal structure solved by Wu et al. [36] has
a 37-residue histidine tag on the N terminus, of which
only two amino acids are ordered and hence visible in the
electron density map.

Table 1
Summary of different crystal forms of GFP currently under study.
Variant Space group Cell dimensions (A) Resolution (A) Form Conditions

Wild-type* P41212 87 x 120 1.9 Dimer MPD, pH 6.8

Nativet C2 93 x 66 x (108) 2.1 Dimer 2M phosphate, prig
Wild-type-His tag~ P6122 77 x 77 x 330 <2.5 Dimer ?
Wild-type P212121 52 x 63 x 69 2.1 Monomer ?
P3122(?) 93 x 9 3 x 188 2.8 ?
S65T* P212121 52 x 6 3 x 71 1.9 Monomer PEG pH8
F64L P3122 90 x 90 x 130 2.3 Dimer
F64L, I167T, K238N *t P6122 9 0 x 9 0 x 130 2.3 Dimer 2M AS pH8.5
P21 93 x 52 x 103 (102) 2.3 Monomer MPD pH8
14132 176 2.5 Dimer 2M AS pH8.5
F64L, $65C, I167T, P6122 90 x 90 x 130 2.1 Dimer (see above)
K238N* 2.3 Dimer (see above)
F64L, Y66H* P6122, 14132 90 x 90 x 130 2.1 Dimer
F64L, Y66H, V163A* P3122 90 x 90 x 130 2.5 Dimer (see above)
14132 2.4 Dimer

*Includes the inadvertent Q80R mutation and an additional alanine at the N terminus, fHas the following sequence differences from the cloned
isoform: R8OG; F100Y; TIO8S; L141M; E172L SHas a 37 residue His-tag at the N terminus (only 0 and -1 positionsseen in the electron density
maps). §Four monomers per asymmetric unit, but not the commonly seen dimer. (a) MA Perozzo, F Yang, GN Phillips, W Ward, K Ward, unpublished
data. AS, ammonium sulfate; MPD, 2-methyl-2,4-pentane diol; PEG, polyethlyene glycol.
826 Proteins
Control of GFP dimerization
Green fluorescent protein can form homodimers both in
solution and in crystals. The KD is approximately 100~t M
as measured by analytical uhracentrifugation (F Yang and
GN Phillips, unpublished data). This weak association is
consistent with the observation that most but not all crystal
forms exist as dimers, and with correlation microscopy
of purified GFP, which reveal a diffusion coefficient
consistent monomers at low protein concentrations [37].
In both the wild-type and native crystal structures, the
crystallographic contacts are all rather tenuous, consisting
of a few amino acids sidechains for each. In contrast,
the dimer symmetry is maintained by extensive contacts
and is similar in at least three structures ([15°',36]; MA
Perozzo, F Yang, GN Phillips, W Ward, K Ward et aL,
unpublished data), and thus is likely to be the source
of the dimerization seen in solution studies. The dimer
contacts are fairly tight and consist of a core of hydrophobic
sidechains from each of the two monomers and a wealth
of hydrophilic contacts. The smaller hydrophobic patch
could conceivably be involved in physiological interactions
with aequorin, as there would be a natural advantage to
close proximity for efficient energy transfer. Fluorescence
changes take place on dimerization [38], and subtle
rearrangements of the fluorophore may also occur upon
dimerization [36].
Control of the dimerization will be important for fluores-
cence resonance energy transfer studies of protein-protein
interactions using GFP [8°,39], as one would not want
to induce association, and hence resonance energy trans-
fer, between the differently colored GFP proteins by
mechanisms other that of the target protein interactions.
The extent of the effect will, of course, depend on the
concentration of GFP.
Conclusions
The 3D structures of GFP have provided a physico-chemi-
cal basis for many of the observed features of the protein,
including stability, fluorophore protection, behavior of
mutants, and dimerization properties. The structures
will also allow directed mutation studies to complement
random mutagenesis and also improve combinatorial
approaches in the production of useful probes in cell and
developmental biology.
Future directions in the study of GFP may well be
in the development of single molecule reporters based
on the transient states seen in some mutants [40].
This would serve to extend the range of colours of
muhireporter funcions, provide better control of the
time required for activation and turnover rates, and the
development of various fluorescence resonance energy
transfer (FRET) assays for protein associations in vivo.
These new chemical tools must be accompanied by
developments in microscopy, including better filter sets
and calibrations [41] but the results will most likely have
a widespread effect in cell and developmental biology.
Acknowledgements
We thank the following for financial support: the Robert A Welch
Foundation; the WM Reck Center for Computational Biology; and the
National Institutes of Health (AR40252 and AR32764).
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