Mutagenesis of V163A and V163G Increases Fluorescence of EGFP
Muhammad Jalal, Eric Montgomery, Ismerai Rodriguez, EJ Crane, and Katy Muzikar
ABSTRACT
Green fluorescent protein (GFP) is an important
tool in a number of biochemical and molecular
biology contexts, such as protein expression and
localization, thanks to its characteristic
fluorescence. Driven by an excited state proton
transfer system which excites a chromophore
embedded in the protein, GFP is tightly protected
from outside forces through a beta barrel structure.
A number of amino acid mutations have been
developed to enhance the fluorescence and
stability of GFP, such as enhanced green fluorescent
protein (EGFP), though most research focuses on
residues immediately surrounding the
chromophore. In this paper, we investigate the
effects mutagenesis of V163 in EGFP, a non-
interacting residue, through mutations V163A and
V163G. Our findings suggest that both mutants
have an increase in fluorescence compared to WT,
though not to the degree indicated by past studies.
INTRODUCTION
The discovery of Green Fluorescent Protein,
originally isolated from Aequores victoria, has
led to a remarkable breakthrough in
investigations of localization and gene
expression within cellular and molecular biology
systems. The 238 amino-acid encoding protein
obtains its fluorescent and spectroscopic
properties from a conjugated -electron
resonance system formed by the oxidation of
the tripeptide chromophore Tyr65-Thr66-
Gly67.1 An excited state proton transfer
mechanism, involving the hydrogen bond
interactions of neighboring amino acid residues
and the chromophore, has been suggested to
contribute to the intensity of fluorescence and
the stability of the protein (Figure 1).2 The
chromophore is imbedded within the interior of
a hydrophobic beta barrel, which protects it
from quenching by polar molecules.
Figure 1. Interacting residues in GFP. The chromophore,
located in the center of the picture, interacts through
hydrogen bonds with a number of surrounding residues,
indicated by their number. This is suggested to be the
mechanism through which fluorescence emerges. Of
particular importance is Q183 (orange), said to interact
with V163. Image obtained from Brejk et al. (1997).
Due to the experimental importance of
GFP, it has become a priority to create mutants
with enhanced properties. Point mutations of
the protein have led to the development of
variants with greater fluorescence, alternative
color emission, and increased stability. A widely
used variant, termed Enhanced Green
Fluorescent Protein (EGFP), involves a mutation
with a chromophore encoding residue and has
been shown to exhibit 3.3 times greater
fluorescence than wild-type GFP.3 Other variants
exploit the hydrogen bonding network through
mutations of chromophore-surrounding
residues, which may be a contributor for both
stability and fluorescence.
Less clear is the importance of residues
more distant from the chromophore. An
interiorly located residue, V163 has been
targeted in a number of successful mutants. A
V163A mutation of EGFP resulted in a 2.7-fold
increase in fluorescence.3 Performing this
mutation in GFP alongside F99S and M153T,
other residues that are distant from the
Figure 2. PyMOL generated images showing mutagenesis of V163A and V163G. (A) The wild-type configuration. V163 (pink) is
located in the interior of the protein, though it does not interact with the chromophore. (B) A potential rotamer of the V163A
mutation. The side chain (pink) decreases, but the protein still points inward. (C) A potential rotamer of the V163G mutation.
Because of the loss of a carbon side chain (pink), the protein no longer points primarily inward.

Table 1. PCR Primer Design for Mutagenesis of V163G in GFP

V163G_Forward 5' tgttatggcgaattttgaagttACCtttgataccgttcttctgtttg 3'
V163G_Reverse 5' caaacagaagaacggtatcaaaGGTaacttcaaaattcgccataaca 3'

studies.4 In thermal stability denaturation

chromophore, resulted in a 42-fold increase in
fluorescence.3 Furthermore, V163A has been
shown to confer enhancement of folding relative
to GFP at an elevated temperature (37C),
suggesting that it may play a role in the stability
of the protein.4
To account for these behaviors, we
investigate the role of three amino acid
properties in the fluorescence and stability of
EGFP: side chain length, hydrophobicity, and -
sheet propensity. Alanine is smaller, less
hydrophobic, and less prone to form -sheets
than valine.4 Our mutation of interest, V163G in
EGFP, involves an amino acid substitution to
glycine, which is yet smaller, less hydrophobic,
and less prone to form -sheets than alanine.4
Based on the exhibited pattern, we
hypothesized that a V163G mutation will result
in greater fluorescence compared to a V163A
mutation. However, theoretical substitutions
reveal the removal of a carbon side chain, as
expected of glycine (Figure 2). This may result in
a decrease in stability of the beta barrel and in
increased exposure to quenching agents,
contributing to diminished fluorescence.
Through fluorimeteric analysis of the
amplified protein fractions of interest, we report
that both V163A and V163G result in an increase
of fluorescence compared to wild-type EGFP,
though not to the degree suggested in previous
assays, no significant difference was observed in
the decrease in fluorescence over most
temperatures among the three variants, save
that V163A was able to retain fluorescence at
the highest temperature while the others were
not able to. In kinetic analysis of thermal
renaturation assays, V163A recovered
fluorescence at a faster rate than the other
mutants. These findings suggest that V163A is a
possible mutant of interest for improving EGFP,
though the specific biochemical mechanisms
behind this are yet to be fully characterized.
METHODS
Primer Design and PCR
Forward and reverse primers corresponding to a
site-directed mutagenesis of V163G were
designed using the Agilent Technologies
QuikChange Primer Design tool (Table 1).
PCR was performed on the p_EGFP2 parent
plasmid using the QuikChange SDM PCR method
as previously described (Table 2, 3). The PCR
product was digested with 1 L DpnI to remove
the parent plasmid and to isolate the amplicon,
and purified using StrataClean resin. A sample of
the PCR product was analyzed through gel
electrophoresis to confirm the presence of a
product.
Transformation of EGFP into XL-10 Gold Cells Sequencing and Analysis
A 100 L aliquot of XL-10 Gold cells was Using the Sanger method, the newly purified
transformed with 10 L of purified PCR product plasmid was sent to be sequenced by Operon.
using a heat-shock method as previously The sequence was analyzed by CLC Sequence
described. The cells were incubated with shaking Viewer through an alignment of EGFP and
in 100 L of SOC media for 45 minutes at 37C, V163G sequence. Pre-existing sequencing data
plated onto a LB-Kanamycin plate, and for the V163A mutation was also compared.
incubated overnight at 37C. Two colonies were
picked and inoculated overnight at 37C in a Transformation of EGFP into BL21(DE3) Cells
shaking LB solution containing 3 mg/mL Four 100 L aliquots of BL21 (DE3) cells were
kanamycin. transformed with 3 L of purified DNA using a
heat-shock method as previously described- 2
Table 2. PCR Reaction Mix Reagents with the V163G mutation, and 2 with the V163A
Reagent and Initial Final mutation. The cells were incubated in a shaking
Concentration Concentration
100 L of SOC media for 30 minutes at 37C,
10x concentrated buffer 1x Buffer
plated onto a LB-Kanamycin plate, and
P28a_EGFP2 Plasmid 0.3 ng/L incubated overnight at 37C. A colony was
(3 ng/L) inoculated overnight at 37C in a shaking LB
Forward Primer (2 M) .2 M solution containing 3 mg/mL kanamycin.

Reverse Primer (2 uM) .2 M Protein Induction and Overexpression

Wild-type EGFP and mutation plasmids
dNTP solution (2.5 mM) .25 mM
corresponding to V163G and V163A were
PfuUltra DNA Polymerase .05U/L overexpressed in BL21(DE3) E. coli cells. Cells
(2.5 U/L) were grown overnight at 37C in 10 mL of LB
media containing 300 g kanamycin. This culture
Table 3. PCR Program was diluted in 500 mL of LB containing 15 mg of
# of Cycles Temperature (C) Time (s) kanamycin and incubated with shaking at 37C.
1 95 120 The OD600 of the cells was measured through
95 20 UV-Vis Spectroscopy until an absorbance of 0.63
30 65 20 was obtained. Following addition of 1M IPTG,
72 360 expression of the T7 promoter was activated
1 72 180 and thereby activated expression of GFP. A 1 mL
4 sample of culture were collected every 30
minutes for 2 hours to analyze induction
Plasmid Growth and Mini-Prep through SDS-PAGE. At the end of this point, the
Plasmids were isolated from a combined remaining cultures were centrifuged, isolated,
solution of both colonies using the Promega and stored at -80C.
WizardPlus mini-prep kit. Quality and
concentration was assessed with a Nanodrop Affinity Purification
spectrophotometer. The obtained plasmid was The cell pellet was suspended in 2 mL Novagen
discarded due to the error in combining the two Bugbuster Master Mix and 50 L of 30 mg/mL
colonies. A new colony was picked, and its PMSF solution. Following incubation on ice and
plasmid extracted through the mini-prep kit, by extraction of the GFP containing supernatant, 50
the lab instructor. Plasmids corresponding to a uL of the supernatant were extracted for the
purified V163A mutation were obtained from a SDS-page gel as crude extract. Metal ion affinity
previous class section. chromatography was used to bind the His tag of
the EGFP protein to a nickel spin column for
purification as previously described. The flow-
through and three 1 mL elution fractions for
each EGFP sample were collected to be analyzed
for the SDS page-gel. The purified protein was
stored at 4C, and the column was regenerated.
SDS Page
SDS gel electrophoresis was used to determine
the induction of EGFP as well as its successful
isolation through affinity purification. The
previously mentioned samples were incubated
in 2X loading buffer and heated for 5 minutes at
95C to be denatured. 10 uL of each sample was
added to each well, along with one lane
containing 10 uL of Precision Plus Protein Dual
Color Ladder, and the gel was allowed to run at
200V for 30 minutes. The gels were stained with
Coomassie Brilliant Blue, left to rock for 10
minutes, and rinsed with dH2O. Following
overnight rocking, the gels were photographed.
Protein Quantification
The two most concentrated fractions were
combined, and their specific concentration was
determined through two methods:
A280
EGFP concentrations were determined using UV-
vis spectrophotometer absorbance readings at
280 nm of 1 mL of each sample. Using Beers law
and the extinction coefficient of EGFP at = 280
nm of 21,890 M-1 cm-1 (as determined by
aromatic residues W1 and Y11), the protein
concentration was determined and converted to
uM.
Bradford Assay
A standard curve of bovine serum albumin (BSA)
absorbance values (determined at 595 nm using
a UV-vis spectrophotometer) was created with
known concentrations of 0, 500, 1000, 1500,
and 2000 ug/mL of protein. The resulting curve
was fitted to a third order polynomial using
Excel. 20 uL of the combined protein for each
EGFP variant was mixed with 1 mL of Bradford
reagent, and the absorbance was determined.
Concentrations in ug/mL were obtained using
the standard curve, and a final concentration in
uM was derived using the dilution factor and an
online weight to molar quantity converter (with
an assumed protein weight of 27 kDA from the
SDS-PAGE gel).
Fluorimetry
Protein fractions were brought to equal
concentration with PBS to ensure proper control
for fluorescence studies. Excitation (490 nm)
and emission (510 nm) values, as well as relative
fluorescence, were determined using a
Beckman-Coulter fluorimeter, with a focus on
the range of fluorescence in between 400 and
600 nm. The gain was manually adjusted to fit
the absorbance within one plot. The points were
graphed through Excel, and local maxima were
used to derive the excitation and emission
values. The relative fluorescence was
determined as the ratio of a samples
fluorescence to the wild type fluorescence.
Stability Assay
The ability of the protein to maintain its
fluorescence intensity was used as a marker of
stability. Two separate experiments were
conducted to analyze this property. Protein
fractions were bound to an Amicon Ultra-0.5
Centrifugal Filter device and recovered in a 0.2M
pH 7.4 phosphate buffer solution (made with
0.2M Na2HPO4 and 0.2M NaH2PO4) to ensure an
ideal environment for stability studies.6
Thermal Denaturation Assay
500 uL of each protein sample was loaded into a
crystal quartz cuvette and placed in a
fluorimeter connected to a single-cell Peltier
accessory. Fluorescence readings in between
400 and 600 nm were taken at 25C and an
excitation wavelength of 490 nm, and the gain
was adjusted to ensure curve fitting. The
samples were next exposed to 37C for 2
minutes using the Peltier accessory, and the
fluorescence was measured. This process was
repeated for increasing temperatures of 50C,
65C, and 80C, and 95C.
Thermal Renaturation Assay
A new 500 uL aliquot of each protein sample
was loaded into a crystal quartz cuvette, and
fluorescence at 25C was determined in a similar
fashion as above. The protein was then exposed
to 65C until ~60% of the fluorescence was left
(fluorescence was measured every 30 seconds
to reach this point). The temperature was
brought down to 25C, and for every minute of
incubation, a fluorescence reading was analyzed
until the protein fluorescence was close to fully
recovered to its initial 25C reading.

Kinetics of renaturation were determined using
the total degree of fluorescence increase from
the initial and final reading, and creating a ratio
evaluating specific increase after each time
point. The data was fitted to a polynomial
equation using Excel.
RESULTS
Confirmation of Product by Gel Electrophoresis
To confirm the presence of a highly amplified
product, a sample of the completed PCR
reaction was loaded and analyzed through gel
electrophoresis. A product of approximately
6000 bp was present in the reaction,
corresponding closely with the length of the
6050 bp p28a_EGFP2 plasmid (Figure 3).
Transformation of V163G EGFP
Following heat-shock transformation of XL-10
Gold cells and overnight incubation, 150
colonies were counted on the LB-Kanamycin
plate. Two colonies were inoculated to confirm
transformation and to amplify the desired
plasmid.
Figure 3. Gel electrophoresis of PCR product (V163 lane)
confirms the presence of a 6000 bp product. For reference,
the length of the p28a_EGFP2 plasmid is 6050 bp.
Mini-prep Product Purity
Following inoculation and purification, the
concentration and purity (using the A260/A280
value) of the obtained plasmid was assessed
using a Nanodrop Spectrophotometer (Table 4).
Table 4. Nanodrop Results for Combined Colony Mini-
prep
Concentration A260/A280
447.5 ng/ L 1.87
Sequencing Analysis
After a new colony was picked and purified
through a mini-prep kit, the DNA was sequenced
using the Sanger method to confirm the
presence of the desired mutation (Figure 4). The
resulting DNA sequence was analyzed against
EGFP and V163A using CLC viewer (Figure 4A).
The triple codon containing the blue codon in
the middle reflected the amino acid.
Figure 4. Sanger Sequencing analysis of single colony mini-prep reveals successful mutagenesis. (A) Alignment analysis using CLC
viewer of EGFP and the reverse compliments of V163A/V163G reveal a single base pair mutation. The triple codon sequences
represented by the blue misalignment, GCT, GGT, and GTT, correspond to alanine, glycine, and valine, respectively. (B)
Chromatogram obtained by Sanger sequencing of V163A and V163G.

Figure 5. SDS-PAGE Gel of Protein Fractions confirms purification of a ~27 kDA protein, matching the literature value weight of
EGFP. (A) Gel images of the V163A mutation. T= corresponds to the various hourly induction points isolated from protein
induction, Crude corresponds to the crude supernatant obtained prior to affinity purification, FT corresponds flow through, M1-
M3 correspond to the mutant EGFP fractions obtained following saturation via elution buffer, and WT1-3 correspond to the wild-
type EGFP fractions obtained similarly. (B) Gel images of the V163G mutation. Notation of the gel is identical to the above.

Confirmation of Protein Purification the presence of other bands, we approximate a

Following confirmation of successful
mutagenesis through sequencing, plasmids
corresponding to each variant were harvested in
BL21(DE3) cells. Protein samples were collected
from various time point inductions of the T7
promoter, as well as eventual elution through
affinity based purification, and analyzed with a
SDS-PAGE gel (Figure 5). Following staining, a
band of approximately 27 kDa appeared after
1.5 hours of induction, as well as in the purified
fractions in both mutant and wild type proteins.
Based on the intensity of the bands, as well as
95% purity rate of wild type, 95% purity rate of
V163G, and a 90% purity rate of V163.
Protein Quantification
The two most concentrated fractions were
combined into one solution. To obtain their
concentrations, two methods were used-
spectroscopy at 280 nm and a Bradford assay
with BSA. A third degree polynomial curve was
fitted to a range of concentrations and
corresponding BSA absorbances (Figure 6).
Concentrations were derived in uM using Beers
law for the 280 nm method and curve derivation
for the Bradford method (Table 5). The results
suggest more mutant protein was extracted
than wild type protein.
Thermal Denaturation
Protein fractions were exposed to incrementally
increasing temperatures for 2 minutes in each
temperature, and the fluorescence driven by a
490 nm excitation beam was determined
through fluorimetry (Figure 8). Each peak was
compared against the initial 25C peak to assess
relative decrease in fluorescence (Table 7). Both
WT and V163 exhibited close to 0% fluorescence
at 95C, while V163A retained 15% of its initial
fluorescence.

Table 7. Relative fluorescence of EGFP over increasing

temperatures.

Figure 6. Bradford Standard Curve of Bovine Serum

Albumin (BSA). The absorbance of five concentrations of
BSA, ranging from 0 to 2000 ug/mL, were fitted to a third
degree polynomial standard curve and used to derive uM
concentrations of protein fractions.

Table 5. Protein concentration for WT EGFP and

mutants using AU280 and Bradford assay. All values
are in uM.
WT V163A V163G
AU280 45 55 60
Bradford 27 45 43
Thermal Renaturation
Fluorimeteric Analysis Protein fractions were exposed to 65C for
Protein samples were recalibrated to a relatively either 2 or 2.5 minutes, resulting in a ~60%
equal concentration and processed through a relative fluorescence from the 25C initial point.
fluorimeter to obtain emission and excitation The temperature was then brought down to
wavelengths, as well as relative fluorescence 25C, and fluorescence was measured every
(Figure 7, Table 6). The wavelength values were minute after for 6 to 7 minutes (Figure 9). V163
relatively consistent among each sample, but was allowed to recover for an additional minute
both mutants had higher relative fluorescence since it was still ~7% away from reaching full
than wild-type EGFP. fluorescence (Table 8).

Table 6. Fluorescent properties of EGFP variants. Kinetics of fluorescence were derived using the
Relative Emission Excitation ratio of fluorescence recovery at each point to
Fluorescence Wavelength Wavelength the total degree of recovery over the 6 or 7
(nm) (nm)
minutes (Figure 10). Through polynomial curve
WT 1 511 489
fitting, more fluorescence of V163A was
V163A 1.7 513 490 recovered at early time points than of the other
V163G 1.5 513 489 variants.
Figure 7. Fluorimetric data of EGFP variants. Dotted lines indicate emission spectra (510 nm), and solid lines indicate excitation
spectra (490 nm). Mutant variants have higher relative fluorescence than wild type EGFP.

Figure 8. Fluorimetric data of thermal denaturation in EGFP variants shows full extinction of fluorescence at 95C in WT and
V163G. Different colored curves reflect different temperatures (as indicated by the legend on the right of each chart). Peaks
reflect emission patterns following an excitation wavelength of 490 nm. (A) WT EGFP. (B) V163A (C) V163G.
Figure 9. Fluorimetric data of thermal renaturation in EGFP variant shows full recovery for V163A and WT following exposure to
denaturation at 65C. Different colored curves reflect different time points (as indicated by the legend on the right of each chart).
Peaks reflect emission patterns following an excitation wavelength of 490 nm. (A) WT EGFP. (B) V163A (C) V163G.

Table 8. Assessment of Renaturation.

 KINETICS OF RECOVERY
V163
100
PERCENT RECOVERY
80
60
40
20
0
1 2 3 4
TIME POINT (MINUTE)
Figure 10. Kinetic assessment of fluorescence recovery after each minute of incubation at 25C shows V163A regenerates
fluorescence faster. Bar graphs indicate cumulative increases in fluorescence after each minute. Dotted curve shows a fitted
polynomial curve for the degree of recovery for each variant.
DISCUSSION
Given the role of V163A in enhancing
fluorescence of EGFP, one hypothesis involved
that a mutation to a residue that was more
hydrophilic, smaller in size, and had an increased
beta sheet propensity would correspond to an
even higher degree of fluorescence. Glycine was
an immediate target for mutagenesis; however,
given that it resulted in the removal of a carbon-
based side chain, we hypothesized that an
alternate effect would be a decrease in the
stability of the protein. To test the effects of
these mutations on fluorescence and stability,
site directed mutagenesis and molecular biology
techniques was used to obtain large amounts of
purified protein.
After engineering primers corresponding to the
induction of the mutations of interests and using
PCR to amplify plasmids containing inducible
versions of EGFP, we used the Nanodrop
method to evaluate the quality of the extract
(Table 4). The A260/A280 ratio of 1.87 suggests
that the plasmid sample was relatively pure.
Sequencing analysis revealed that the desired
mutations were obtained (Figure 4).
V163A V163G
5 6
Plasmids were harvested in BL21(DE3) cells to
induce overexpression. SDS-PAGE gel lanes
corresponding to points after the T7 promoter
was induced reveal that the protein began to
emerge around 1 hour and was expressed more
and more after each following point (Figure 5).
Following extraction of protein samples from the
cells, the crude extract was run through a nickel
based chromatography column, and elution
fractions were analyzed through SDS-PAGE to
confirm purity. The elution lanes confirm the
presence of EGFP in all of the variants, and an
approximation of purity (using the presence of
other bands) suggests that the elution fractions
were 90-95% EGFP (Figure 5).
The protein fractions were quantified through
two methods: spectroscopy at 280 nm and a
Bradford assay. Our results indicate that more
mutant protein was extracted, though this may
have been a non-characteristic feature given the
variation from one cell culture to the next (Table
5). The concentration of each protein was made
the same through PBS dilution, and the
fluorescence was analyzed through fluorimetery
(Figure 7).
Both mutants had similar wavelength properties
of emission and excitation as WT-EGFP,
suggesting that the color of fluorescence did not
significantly change following mutagenesis. The
values obtained were close to literature values
of EGFP.4 V163A was initially found to be more
fluorescent (by 13.3%) than V163G. Relative
fluorescence studies, however, revealed that
both mutants were substantially more
fluorescent than WT-EGFP- 50 to 70% more
fluorescent (Table 6). However, this is also
significantly lower than the 2.7-fold increase
reported in the V163A mutant of EGFP in other
studies.4 This is best explained through the
variance in environment for our protein samples
compared to that of others. Indeed, upon
performing stability assays using a different
buffer system, the mutants had identical
fluorescence to each other prior to exposure to
elevated heat (data not shown).
Because V163A was also indicated as an
enhancing residue for protein folding in elevated
temperatures, we were interested in assessing
the thermal stability of both mutants. Proteins
were reconstituted in a pH 7.4 phosphate
buffer, which was chosen as it was also used
effectively in other thermal stability assays.6
Thermal denaturation and renaturation assays
were conducted as previously described. The
preservation of fluorescence after thermal
denaturation at increasingly larger temperatures
did not seem to differ significantly from one
protein to the other; however, a noteworthy
finding was that V163A retained some
fluorescence at 95C while the others were
completely extinguished (Figure 8, Table 7). On
an absolute magnitude, V163A preserved more
fluorescence at the varying temperatures than
the other mutants save one exception, but
duplicate or triplicate analysis would be needed
to confirm if this is a significant finding. The
thermal degradation studies indicate that V163A
confers increased protection of the fluorescent
signal at extremely high temperatures, whereas
V163G does not.
A thermal renaturation assay was performed to
characterize a different feature of the protein-
its ability to refold following thermal
denaturation and to restore its fluorescent
quality. Two hypotheses were implicated: that
V163A would confer faster renaturation given its
conference of thermal stability as previously
shown, or that V163G would be faster due to
the increased beta-sheet propensity of glycine.
While both WT and V163 recovered to full or
close to full fluorescence after 6 minutes, V163G
was further away from full fluorescence than the
others after a longer period of recovery at 7
minutes (Table 8). To better qualify the rate of
recovery over time, a polynomial curve was
fitted to the compounding nature of recovery
over time (Figure 10) among each variant. In
every time point, V163A recovered fluorescence
faster than the other two; V163G did not
recover much fluorescence after the first
minute.
Nonetheless, these thermal investigations must
be taken with considerable caution given the
number of confounding factors present in the
methodology. An assumption made was that the
Peltier machine would increase or decrease in
temperature at a consistent rate from sample to
sample; if this assumption is inaccurate, it would
severely undermine these findings. Given the
amount of human interaction associated with
timing, measuring, and switching the
temperature, it is very likely that some data,
such as the kinetic recovery data, is more a
measure of these forces than anything inherent
to the mutations. Developing a better method
would be a priority in continuing these
investigations. The use of other assays, such as a
pH assay, a quenching essay, and folding assays,
would enhance understanding of stability.7
Given the initial premise of relating amino acid
properties to the nature of fluorescence and
stability in GFP and EGFP, there remain a
number of variables which likely complicate this
parallel. Rather than seeing each residue as a
part which contributes to the entirety of the
protein, it is important to consider how each
residue interacts with others to carry out its
role. While papers have suggested that V163
interacts with Q1832, characterization of amino
acid interactions in GFP remain unclear,
particularly for non-chromophore interacting
residues. Although some work has been done to
elucidate these relationships, using X-ray
crystallography to further understand the
specifics the hydrogen bonding network would
enhance analysis of these investigations.8 After
this is established, the next logical step would be
to continue site-directed mutagenesis of other
amino acids to see if any trends can be
identified.
Ultimately, this work shows the importance of
non-chromophore residues in significantly
governing the properties of GFP. V163A in
particular shows promise as an enhanced
version of EGFP, showing increased fluorescence
and thermal stability than the wild-type variant.
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