Binding and signaling of surface-immobilized
reagentless fluorescent biosensors derived from
periplasmic binding proteins
ROBERT M. DE LORIMIER, YAJI TIAN, AND HOMME W. HELLINGA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA
(RECEIVED March 31, 2006; FINAL REVISION May 18, 2006; ACCEPTED May 18, 2006)
Development of biosensor devices typically requires incorporation of the molecular recognition element
into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal
transducing protein directly on a solid surface. Here we compare the effects of two direct immo-
bilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent
biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose
binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respec-
tively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified
lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces
derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand
binding and signal transduction but with adversely affected affinities and signal amplitudes for the
thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically
oriented layer to maleimide-derivatized plates using a His2Cys2zinc finger domain fused at either their
N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity
and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as
demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with
the immobilized proteins are on the order of seconds.
Keywords: biosensor; surface immobilization; periplasmic binding protein; fluorescence; zinc finger;
Solute binding members of the periplasmic binding pro-
tein (PBP) superfamily have been intensively studied as
receptors for sensor applications (Hellinga and Marvin
1998). These proteins exhibit high specificity and affinity
for their natural cognate ligands and can be designed to
bind nonnatural ligands (Marvin and Hellinga 2001;
Looger et al. 2003; Allert et al. 2004). Ligand binding
is accompanied by conformational changes in the protein,
which can be linked to changes affecting site-specifically-
attached fluorophores, thereby transducing binding into
a fluorescent signal (Gilardi et al. 1994; Marvin et al.
1997; de Lorimier et al. 2002). This engineered reagent-
less sensing mechanism is potentially well suited for real-
time sensing applications.
Development of sensor devices requires incorporation
of sensing proteins into a detector element by encapsu-
lation or surface immobilization on a suitable material for
interfacing with detectors. Here we describe studies for
the immobilization of engineered fluorescent signal trans-
ducing PBPs. Reagentless sensing systems may have the
signal transducing protein separated from the fluid sam-
ple by a diffusion barrier. For example, the protein may
be entrapped in a porous material through which small
molecules diffuse to reach equilibrium with the immo-
bilized receptor (Topoglidis et al. 1998; Alarcon et al.
Reprint requests to: Homme W. Hellinga, Department of Biochem-
istry, Duke University Medical Center, Box 3711, Durham, NC 27710,
USA; e-mail: email@example.com; fax: (919) 684-8885.
Article published online ahead of print. Article and publication date
are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062261606.
Protein Science (2006), 15:1936–1944. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2006 The Protein Society
2005). Alternatively, the receptor protein is directly
linked to a surface and exposed to the fluid sample
without an intervening barrier. This arrangement poten-
tially reduces the time for an analyte molecule to reach
equilibrium with the receptor, which is desirable for moni-
toring real-time analyte concentration fluctuations. PBPs
linked directly to surfaces exhibit signal transduction by
surface plasmon resonance when covalently attached to
carboxymethyl dextran chips (Hsieh et al. 2004) or directly
to gold (Luck et al. 2003). We are unaware of reports
describing signal transduction by fluorophore-labeled PBPs
linked directly to surfaces. The present studies were under-
taken to explore such schemes. We examined whether the
proteins could be attached to surfaces and, if so, whether
ligand-specific fluorescence signal transduction is retained.
The degrees of signal transduction and ligand affinity for
different immobilization methods were also examined.
Other critical questions that were pursued concern the re-
versibility of the response to ligand binding, and the useful
lifetime of the surface as measured by the maximum
number of consecutive cycles of binding and elution.
The proteins used in this study were thermostable sugar
binding proteins cloned from the genomes of two ther-
mophilic bacteria: a ribose binding protein from Thermo-
anaerobacter tengcongensis (TteRBP) and a glucose
binding protein from Thermotoga maritima (TmGBP).
These proteins have been characterized with regard to
thermostability, ligand binding, crystal structure, and
fluorescence signal transduction in solution (S. Rizk,
Y. Tian, J. Qiu, and H.W. Hellinga, unpubl.; Y. Tian,
A. Changela, M.J. Cuneo, B. Ho ¨cker, L.S. Beese, and
H.W. Hellinga, unpubl.). Both proteins exhibit sequence
and structural homology with other periplasmic mono-
saccharide binding proteins, facilitating the selection of
residues for attachment of fluorophores based on obser-
vations from studies on other monosaccharide binding
proteins (Marvin and Hellinga 1998; de Lorimier et al.
2002) and other classes of PBP (Marvin et al. 1997; Marvin
and Hellinga 2001; de Lorimier et al. 2002). Because of their
thermostability, these and other such proteins offer potential
advantages for use in harsh environments or as scaffolds for
designing novel binding sites.
We examined surface immobilization and signal trans-
duction using 96-well microtiter plates preactivated with
various linking functions, to which fluorescent signal-
transducing periplasmic binding protein was attached and
from which fluorescence emission was quantified using
a microplate reader. This system allows rapid measure-
ments of samples under multiple treatments.
Two general modes of protein attachment to microplate
wells were examined. In the semispecific immobilization
mode, amine groups on the protein were covalently
conjugated with biotin or thiol groups. (We use the term
semispecific rather than nonspecific immobilization be-
cause the latter implies physisorption through indetermi-
nate interactions.) This conjugate was immobilized by
reacting with either streptavidin-coated microplate wells
(for biotin conjugation) or maleimide-derivatized wells
(for thiol conjugation). Conjugation reactions were con-
trolled to limit the extent of biotin or thiol addition to
protein (averaging between two and three adducts per pro-
tein), but because of the large number of primary amines
in TteRBP (29 lysines and the N terminus), there are
many possible permutations of the conjugation pattern.
In the specific immobilization mode of protein attach-
ment, the signal transduction protein was expressed as
a fusion to a zinc finger domain at either its N or C ter-
minus. After labeling the unprotected thiol (TteRBP
Cys168 or TmGBP Cys13) with a thiol-reactive fluoro-
phore, the fusion protein was prepared for covalent attach-
ment to microplate wells by removing the coordinated
zinc from the Cys2His2zinc finger and reducing the result-
ing zinc finger disulfide to thiols, which then reacted with
maleimide groups in the wells of the plate.
Semispecific immobilization via biotin
TteRBP-D168C labeled with Cy3 or Cy5 and conjugated
with PEO4-biotin was attached to streptavidin-coated
wells in microplates. The amount of protein bound per
well was estimated by fluorescence intensity, calibrated
with a serial dilution of the unconjugated labeled protein
in solution. Typically 2–3 pmol of biotin-conjugated protein
was bound per well, quantified by absolute fluorescence
intensity. Two possible sources of error in this estimate may
derive from (1) a difference in the efficiency with which
the instrument optics collects fluorescence from the bottom
of wells compared to the full volume of the wells, and (2)
a difference in quantum yield of the fluorophore attached to
protein in solution compared with that attached to immobi-
lized protein. For comparison, the maximum biotin binding
capacity of the wells as stated by the suppliers is 125
(Pierce) or 300 (Sigma) pmol, but these figures are for small
standards such as biotin-fluorescein and are not expected to
be achieved for larger molecules such as biotin-conjugated
proteins. The observed 2–3 pmol of protein per well is con-
sistent with the expected maximum amount of 14 pmol,
assuming a completely packed monolayer of PBP having
a molecular diameter of 30 A˚, a diameter of 4 mm for the
microtiter well, and 4 mm for the height of the active sur-
face on the wall of a well.
Biotin-conjugated TteRBP-D168C labeled with Cy3 or
Cy5 was immobilized on streptavidin-coated wells and
titrated with ribose. Fitting of the titration curve to a simple
two-state hyperbolic binding isotherm produced a parameter
Surface-immobilized fluorescent biosensors
for ligand binding affinity (Kd) and a parameter for signal
transduction (DFmax), as illustrated in Figure 1. These
parameters were derived for the protein titrated in solution
and for unconjugated TteRBP-D168C, and compared to
reveal the effects of conjugation and surface immobilization
on ligand affinity and signal transduction, as summarized in
Table 1. We observed that conjugation with PEO4-biotin had
little effect on Kdor DFmaxfor either the Cy3- or Cy5-
labeled protein. Upon immobilization of biotin-conjugated
TteRBP, Kdand DFmaxdecrease about twofold for the Cy5-
labeled protein but change only slightly for the Cy3-labeled
protein. We conclude that semispecific surface immobili-
zation by the biotin-streptavidin interaction generally pre-
serves the ability of TteRBP to bind ligand and transduce
a fluorescent signal.
Semispecific immobilization via thiols
TteRBP-D168C labeled with Cy3 or Cy5 and conjugated
with 2-iminothiolane was titrated both in solution and
immobilized on maleimide-activated plates and compared
with unconjugated protein (Table 1). Conjugation had
slight effect on Kdand DFmaxin solution, but immobili-
zation resulted in greatly diminished DFmax for both
fluorophores studied (Table 1).
A defined single attachment point from a protein to
a surface can be achieved through the use of particular
peptide sequences fused to a terminus of the protein. Here
we use a zinc finger domain containing two cysteine
thiols coordinated to a zinc ion. Previously we have
demonstrated that these two cysteines can be functional-
ized independently of other cysteine residues in the fusion
partner by removing the zinc ion, reducing the resulting
disulfide, and reacting with a thiol-reactive moiety (Smith
et al. 2005).
Genes encoding zinc finger fusions at the N or C
terminus of TteRBP-D168C and TmGBP-Y13C were
constructed and expressed in Escherichia coli. TteRBP-
CZif and TteRBP-NZif labeled with Cy5 at Cys168 were
immobilized to maleimide-activated microtiter wells to
quantify the efficiency of binding by fluorescence in-
tensity, calibrated with a serial dilution of the nonimmo-
bilized protein in solution. Typically 1.5–2.5 pmol of
bound protein per well was estimated by this method. For
comparison, the stated binding capacity of the wells is
100–150 pmol, using sulfhydryl-containing peptides as
the standard. As for the biotin-immobilized proteins
described above, the observed 1.5–2.5 pmol of protein
bound per well is consistent with the estimated maximum
capacity for a protein of this size.
Figure 1. Ribose titration of biotin-conjugated TteRBP-168C-Cy5. The
protein was titrated in solution (squares/dashed line, right axis) or immobi-
lized on streptavidin-coated microtiter wells (circles/solid line, left axis). For
each titration, the displayed curve is the best fit to a two-state binding model
(Marvin et al. 1997). We observe an increased affinity for ribose on the
surface (Kd¼ 85 nM), compared with the protein in solution (Kd¼ 360 nM).
Data are presented as the average of at least three measurements.
Table 1. Signal transduction and ligand affinity in fluorophore-labeled TteRBP
N.A., not applicable; (a), determined from a single experiment.
aValues have standard deviation in parentheses.
de Lorimier et al.
Protein Science, vol. 15
TteRBP-CZif and -NZif labeled with Cy3 or Cy5 were
titrated in solution with ribose to determine the effect of
the zinc finger domain on ligand affinity and signal
transduction (Table 1). For both fluorophores, little
difference in Kd or DFmax between the nonfusion and
the zinc finger fusion was observed (Table 1). When
immobilized to maleimide-activated plates, Cy5-labeled
TteRBP-CZif and TteRBP-NZif showed striking and
reproducible differences with the nonimmobilized pro-
teins, in both Kdand DFmax(Fig. 2). For both proteins, the
Kdimproved five- to sixfold, while DFmaxincreased two-
to fourfold. In contrast to Cy5-labeled protein, Cy3-
labeled TteRBP-CZif showed minor differences in ligand
affinity and signal transduction between the immobilized
protein and the protein in solution (Table 1). Hence, depend-
ing on the fluorophore, specific immobilization preserves or
enhances ligand affinity and signal transduction.
For all derivatives of TteRBP the signal transduction
responses were specific to D-(?) ribose. For example,
titration with D-(+) glucose over a similar range of con-
centrations failed to elicit a change in fluorescence emission
for immobilized TteRBP-CZif labeled with Cy3 (Fig. 3).
Specific immobilization was also tested with TmGBP-
CZif and TmGBP-NZif, labeled with Cy5 at Cys13. This
combination of fluorophore and amino acid residue was
studied because it causes the affinity of TmGBP for
glucose (Kd¼ 10 mM) to be in a range close to that of
the concentration of glucose (5 mM) in human blood
(Burtis and Ashwood 1994), a necessary parameter for
use as a medical glucose sensor. TmGBP-CZif and -NZif
were titrated in solution with glucose to determine the
effect of the zinc finger domain on ligand affinity and
signal transduction. For both zinc finger fusion proteins,
no large differences in DFmaxwere observed compared
with the nonfusion proteins. The zinc finger fusion had
little effect on the affinity of TmGBP-NZif for glucose,
but in the case of TmGBP-CZif, it was correlated with an
approximately twofold decrease in affinity (Table 2).
Both zinc finger fusion derivatives of TmGBP couple
to maleimide-activated microtiter wells to approximately
the same degree (;2 pmol/well) as did the TteRBP
fusions. Compared with solution titration, specifically
immobilized TmGBP-CZif and -NZif both exhibit about
a twofold decrease in DFmaxand an increase in glucose
affinity of two- or threefold (Table 2).
Reversibility of ligand binding to immobilized protein
The reversibility of ligand binding was examined for both
semispecifically (biotin) and specifically (zinc finger)
immobilized TteRBP-D168C-Cy5 and for specifically
immobilized TmGBP-Y13C-Cy5. Arrays of wells con-
taining immobilized protein were titrated with ligand, as
described above, and then repeatedly rinsed with buffer to
remove ligand, as monitored by recording fluorescence
emission after each rinse. Elution of ligand was assumed
to reach completion when the fluorescence from each
well showed no significant change between rinses. The
cycle of titration and rinsing was repeated up to six times.
Comparison of titration curves reveals that immobi-
lized receptor maintains function over multiple cycles of
binding and elution. Figure 4A shows titration data for the
first and the fifth such cycle for maleimide-immobilized
TmGBP-CZif. Among five titrations, values for Kdwere
6.8 6 1.7 mM for TmGBP-CZif and 9.3 6 1.7 mM for
TmGBP-NZif. No correlation was observed between
Kdand titration number, suggesting that ligand affinity
is unaffected by multiple titration treatments. Values for
DFmaxwere ?25 6 1% for TmGBP-CZif, and ?24 6 2%
Figure 2. Ribose titration of TteRBP-D168C-Cy5 fused to a zinc finger
QNK domain and immobilized on maleimide-derivatized microtiter wells.
(Circles/dashed line) N-terminal fusion (TteRBP-NZif); (squares/solid
line) C-terminal fusion (TteRBP-CZif). The Kd values of the N- and
C-terminal fusions are 51 and 81 nM, respectively. Values of DFmaxare
?79% and ?41%, respectively. Data are presented as the average of at
least three measurements.
Figure 3. Immobilized TteRBP-CZif-Cy3 binds ribose specifically. Pro-
tein immobilized in maleimide-activated wells was titrated with D-(?)
ribose (circles/dashed line) or D-(+) glucose (squares/solid line). Data are
presented as the average of at least three measurements.
Surface-immobilized fluorescent biosensors
for TmGBP-NZif, also with no correlation to titration
number. Thus TmGBP specifically immobilized on a mal-
eimide-derivatized surface exhibits reproducible ligand
affinity and signal transduction over several cycles of
titration and elution. Similar results were observed for
maleimide-immobilized TteRBP-CZif-Cy3 over six cycles
of titration (Fig. 4B) and for biotin-conjugated TteRBP-
D168C-Cy5 immobilized to streptavidin-coated microtiter
wells, over two cycles (data not shown). We conclude that
both methods of immobilization retain reversible ligand
The on- and off-rates for glucose with respect to immo-
bilized TmGBP were within the mixing time for adding or
eluting glucose (<30 sec), as judged by a constant
fluorescence intensity between the first and subsequent
readings of a plate after adding glucose or rinsing with
buffer. The on-rate for ribose with respect to immobilized
TteRBP is also within these limits. However, wells
containing immobilized TteRBP-Cy5 had to be rinsed
several times over many minutes or hours to reach
fluorescence levels for the apoprotein. We estimated the
off-rate of ribose from immobilized TteRBP-CZif-Cy5 by
measuring the time dependence of fluorescence after
removing ribose solution from wells containing protein.
The data were fit to a first-order exponential rate model,
from which a pseudo-first-order rate constant was de-
rived. Figure 5 shows typical data and a fit for an
experiment in which the plate was rinsed four times over
2 min, and then fluorescence was recorded every 60 sec
for a duration of 30 min, shaking for 45 sec between each
reading. The derived rate constant is 3.5 3 10?3sec?1,
about 0.2% of the off-rates for arabinose and glucose
binding proteins in solution (Miller et al. 1983). Extrap-
olation of fluorescence to the time of the initial rinse
gives 54%, an estimate of the fraction of the fluorescence
that recovers within a few seconds of rinsing. Therefore
;46% of the protein population exhibits a slow change in
fluorescence. An estimate of the expected off-rate for
ribose from immobilized TteRBP-CZif-Cy5 was made
using the apparent Kdof 37 nM (Table 1) and a typical
on-rate of 2 3 107M?1sec?1found for other mono-
saccharide binding proteins (Miller et al. 1983). Assum-
ing the relation Kd¼ koff/kon, the expected off-rate for
ribose from immobilized TteRBP-CZif-Cy5 is 0.7 sec?1,
about 200-fold more rapid than that estimated for the
slowly recovering population.
Table 2. Signal transduction and ligand affinity in fluorophore-labeled TmGBP
N.A., not applicable.
aValues have standard deviation in parentheses.
Figure 4. Successive cycles of titration and washing of zinc finger
immobilized sugar binding proteins. (A) Cy5-labeled TmGBP-Y13C-CZif
was cycled five times with glucose. The first (circles/solid line) and last
(open squares/dashed line) titration curves are shown, having Kdvalues of
4.6 and 8.0 mM, respectively. The average Kdfor all five titrations was
6.8 6 1.7 mM. (B) Cy3-labeled TteRBP-D168C-CZif was cycled six
times with ribose; the first (circles/solid line) and last (open squares/dashed
line) titration curves have Kdvalues of 15 and 42 nM, respectively. The
average Kdfor all six titrations was 34 6 19 nM, and the average DFmax
was ?17 6 1%. Data are presented as the average of at least three
de Lorimier et al.
Protein Science, vol. 15
The choice of a method for confining a signal transducing
receptor molecule to a surface or a volume element for
applications in sensing depends in part on the sampling
method and on limitations of the signal transduction
mechanism. Topoglidis et al. (1998) observed that fluo-
rophore-labeled maltose binding protein displayed atten-
uated signal transduction (about fourfold less), but
relatively constant affinity, when confined in TiO2gel.
However, Alarcon et al. (2005) found enhanced signal
transduction (two- to threefold higher) for fluorophore-
labeled glucose binding protein when entrapped in a glyc-
erol modified silicate condensate sol-gel. We have exam-
ined schemes for attaching receptors directly to solid
surfaces to construct chemo-responsive surfaces that are
in rapid equilibrium with solutes, which may be appro-
priate for applications that require observing at closely
spaced time intervals.
We observed that surface-immobilized fluorophore-
tagged PBPs retain their response to ligand binding. We
found that structural details of the immobilization mech-
anism can significantly affect ligand affinity and signal
transduction. Immobilization of semispecifically labeled
biotin conjugates to a streptavidin-coated surface per-
turbed ligand affinity and signal transduction by less than
2-iminothiolane and attachment to a maleimide-activated
surface causes a three- to sixfold decrease in signal trans-
duction. Orientation-specific immobilization using zinc
finger fusions either retained or enhanced ligand affinity
(two- to sixfold) and signal amplitude (two- to fourfold),
and is therefore the most reliable and robust method.
We examined the ligand binding reversibility in the
semispecifically biotinylated and specifically immobi-
lized proteins and found that ligand affinity and signal
transduction are reproducible over at least five or six
cycles of ligand loading and elution. This is a critical
requirement for continuous or repeated use of a sensor.
The rate at which the ligand binding equilibrium is
established is also an important factor for real-time
sensing: For use in a continuous flow biosensor, ligand-
exchange kinetics must be matched to the timescale of
fluctuations in ligand concentration. Ligand exchange
observed for immobilized PBPs in this study was com-
plete within 135 sec. The exception is immobilized
TteRBP, where the ligand off-rate is characterized by
two populations, one (;54%) having a rapid off-rate
(koff> 0.1 sec) and the other (;46%) with an estimated
koffof 3.5 3 10?3sec?1. The off-rate for other mono-
saccharides, such as arabinose and glucose, from their
respective binding proteins in solution is ;1.5 sec?1
(Miller et al. 1983).
Taken together, our results demonstrate that thermo-
stable PBPs that have been engineered to function as
reagentless fluorescent biosensors retain their ability to
signal when immobilized on surfaces and have reasonably
rapid ligand-exchange kinetics. The resulting chemo-
responsive surface potentially could be used to construct
optical-based sensors for the monitoring of analyte con-
centrations that fluctuate over a period of seconds.
Further development of surface-immobilized signal-
transducing proteins for incorporation into sensor devices
will require an assessment of the heterogeneity of the pro-
tein population with respect to ligand-exchange kinetics.
Also, the upper limits of iterative cycling will need to be
explored. Thermostable proteins such as used in this
study may offer advantages for robustness of ligand
affinity and signal transduction with respect to multiple
cycles of use. They may also be useful as scaffolds for
designing novel binding functions, as structural stability
may be better retained than for designed proteins derived
from scaffolds from mesophilic organisms.
Materials and methods
Gene construction and expression
The gene encoding a ribose binding protein from the thermo-
philic bacterium TteRBP was cloned from the genome (Bao
et al. 2002) into the plasmid vector pET21a (S. Rizk, Y. Tian,
J. Qiu, and H.W. Hellinga, unpubl.). The gene has been modified
from the wild-type sequence in three ways: (1) 20 residues
comprising the presumptive signal peptide at the N terminus have
been removed and replaced with a Met codon for translation
initiation; (2) Asp168 has been replaced with Cys for fluorophore
attachment; and (3) a six-His tag preceded by a GlySer linker
has been fused to the C terminus for protein purification using
immobilized metal affinity chromatography.
Figure 5. Time-dependence of the recovery of fluorescence from immo-
bilized TteRBP-CZif-Cy5. The fluorescence ratio prior to the ribose
elution step was 1.0. At t ¼ 0 the immobilized protein was rinsed to elute
ribose, and fluorescence was first recorded at t ¼ 2.25 min. The curve is
the best fit with a first-order exponential model to the slow phase, yielding
the fit parameters of koff¼ 3.5 3 10?3sec?1and fluorescence ratios of
1.40 at t ¼ 0 min and 1.76 at t ¼ a. The ratio of 1.40 is 54% of the range
from 1.0 to 1.76, representing the fast phase of exchange that is complete
within a few seconds of rinsing.
Surface-immobilized fluorescent biosensors
The gene encoding a glucose binding protein from the
thermophilic bacterium TmGBP was cloned from the genome
(Nelson et al. 1999) into the plasmid vector pET21a (Y. Tian, A.
Changela, M.J. Cuneo, B. Ho ¨cker, L.S. Beese, and H.W.
Hellinga, unpubl.). The coding sequence has been modified in
three ways: (1) replacing the first 31 residues (signal peptide) with
a Met codon for translation initiation, (2) mutating Tyr13 to Cys for
fluorophore attachment, and (3) fusing to the C-terminal Phe304
residue a GlySer linker followed by a six-His tag for protein
purification using immobilized metal affinity chromatography.
Genes encoding TteRBP-D168C or TmGBP-Y13C fused to
a 33-residue His2Cys2zinc finger domain (ZifQNK) at either the
N or C termini of the former two proteins were constructed as
described (Smith et al. 2005). The gene containing ZifQNK
fused to the N terminus of TteRBP-D168C (TteRBP-NZif) has
a two-residue linker of GlySer between the two domains and has
the same C-terminal His tag as above. For fusion to the N
terminus of TteRBP-D168C, a 109-bp PCR product containing
ZifQNK was constructed using the following two partially
complementary oligonucleotide primers: 1 (sense), CACCATG
CACATCAGAACAAGAAGGGTTCT; 2 (antisense), AGAACCC
CGATCGAGAGAATGATTT. The 59 end of the sense primer
contains the sequence CACC for cloning into the vector pET101/
D-TOPO (Invitrogen) according the instructions of the supplier.
This 109-bp fragment was fused to TteRBP using an oligonucle-
otide primer having overlapping homology with the C terminus of
ZifQNK and the N terminus of TteRPB. The resulting 1-kb
fragment was cloned into pET101/D-TOPO. The gene containing
ZifQNK fused to the C terminus of TteRBP-D168C (TteRBP-
CZif) had a GlyGlySer linker between the C-terminal residue
(Gln278) of TteRBP and Thr2 of ZifQNK; Met1 of the latter being
deleted. The C terminus of this ZifQNK domain was linked by
GlySer to a six-His tag. For fusion to the C terminus of TteRBP-
D168C, a 124-bp PCR product containing ZifQNK was constructed
using the following four partially complementary oligonucleotide
primers: 1 (sense), ACAGGTGAGAAACCGTACAAGTGCCCG
GAGTGTGGCAAATCATTC; 2 (sense), ATCGGACCATCTATC
CACACTCCGGG; and 4 (antisense), GTTAATGGTGGTGGTGA
TGATGAGAACCCTTCTTGTTCTGATGTGTCC. Primer 4 en-
codes a six-His tag linked to the C terminus of ZifQNK by GlySer.
The resulting 124-bp fragment was fused to TteRBP using an
oligonucleotide primer having overlapping homology with the
N terminus of ZifQNK and the C terminus of TteRPB. The result-
ing 1-kb fragment was cloned into pET21a.
The gene containing ZifQNK was fused to the N terminus of
TmGBP-Y13C with similar method mentioned above, with
a two-residue linker of GlySer between the two domains, and
has the same C-terminal His tag as above. As done with TteRBP,
the gene containing ZifQNK was also fused to the C terminus of
TmGBP-Y13C with a GlyGlySer linker between the C-terminal
residue (Phe304) of TmGBP and Thr2 of ZifQNK, with Met1 of
the latter being deleted. The C terminus of this ZifQNK domain
is linked by GlySer to a six-His tag. The expression vector for
both TmGBP-CZif and TmGBP-NZif is pET21a.
Plasmids were transformed into the E. coli strain Rosetta-
gami (DE3; Novagen), and transformants were grown in Hyper
Broth medium (Athena Enzyme Systems) at 37°C. Protein ex-
pression was induced by adding isopropyl-b-D-thiogalactoside
to the cultures when OD600reached ;0.6 units. After shaking
overnight, the culture was centrifuged, and the cell pellet was
suspended in a solution of 500 mM NaCl, 10 mM imidazole, and
20 mM MOPS (pH 7.8) and stored frozen at ?80°C. The cell
suspension was thawed, chilled on ice, and disrupted by
sonication with a Sonifier 250 (Branson) and narrow-tip probe,
using six cycles of 30 bursts each at 50% duty cycle and an
Output Control setting of 5. Five minutes of sample cooling
followed each cycle. The cell lysate was centrifuged for 30 min
in the cold at 30,000 rcf, and the supernatant was collected and
heated in a water bath for 15 min at 65°C. The resulting pre-
cipitate was pelleted by centrifugation, and the cleared super-
natant, containing the thermostable PBP, was loaded on a 3-mL
column of Chelating Sepharose Fast Flow (Amersham Bio-
sciences) preloaded with Ni2+. The column was washed with
40 mL of 500 mM NaCl, 10 mM imidazole, and 20 mM MOPS
(pH 7.8). Subsequent 20 mL washes contained the same buffer
with increasing concentrations of imidazole: 25, 50, 75, 100,
200, and 400 mM. Fractions were collected for each increment
of imidazole concentration and analyzed by SDS-PAGE. Suit-
able fractions were concentrated to 10 mL and further fraction-
ated by gel filtration on a column of Superdex 75 HiLoad
(Amersham Biosciences). Protein-containing fractions were
concentrated and dialyzed into MOPS-buffered saline (MBS;
100 mM NaCl, 20 mM MOPS at pH 7.0). Stock solutions of
ZifQNK-fusion proteins also contained ZnCl2at 100 mM.
Proteins were prepared for labeling by first adding Tris[2-car-
boxyethyl] phosphine to 300 mM and incubating at room
temperature for 10–30 min to reduce any disulfide bonds. This
treatment does not affect the zinc-coordinated ZifQNK domain.
Next, the thiol-reactive dyes Cy3- or Cy5-maleimide (Amer-
sham Biosciences) were added in molar excess according to the
manufacturer’s protocol. After 4 h of reaction at room temper-
ature in the dark, the solution was fractionated by gel filtration
to separate unincorporated dye from labeled protein. The
relative amounts of labeled and unlabeled protein were esti-
mated by MALDI-TOF mass spectrometry; all preparations had
at least 75% labeling efficiency. No species were detected in
ZifQNK fusions that would correspond to additional labeling of
the zinc finger Cys residues.
TteRBP-D168C labeled with Cy3 or Cy5 was conjugated to
biotin using the reagent NHS-PEO4-biotin (Pierce), following
the manufacturer’s protocol. The mass distribution of PEO4-
biotin moieties on the protein was assessed by MALDI-TOF
mass spectrometry. The mean number of biotins per protein
ranged from two to three. A typical distribution of biotin
adducts per protein, having an average of 2.0, is as follows
(adducts, fraction of protein): (0, 0.15), (1, 0.25), (2, 0.25),
(3, 0.19), (4, 0.11), (5, 0.05). Biotin-conjugated protein was
diluted to 1.5 mM and added in 100 mL to each well of a 96-well
streptavidin-coated microplate. Two sources of plate yielded
similar results: Reacti-Bind streptavidin-coated high binding
capacity plates (Pierce) and SigmaScreen streptavidin HC coated
plates. Plates were shaken for 4 h in the dark at room temperature,
after which the wells were rinsed four times with MBS.
Fluorophore-labeled TteRBP-D168C was decorated with
sulfhydryl groups at lysine residues by conjugation with the
reagent 2-iminothiolane (Traut’s reagent) following the manu-
facturer’s protocol (Pierce). The mass distribution of adduct on
de Lorimier et al.
Protein Science, vol. 15
the protein was assessed by MALDI-TOF mass spectrometry
and found to average 2.4–2.7 adducts per protein molecule. The
distribution of 2-iminothiolane adducts per protein molecule
was not quantified because the smaller mass of the 2-imino-
thiolane adduct (137 amu) led to poor resolution of adduct peaks
compared with PEO4-biotin adducts (474 amu).
Fluorophore-labeled zinc finger fusion proteins were prepared
for immobilization according to the method of Smith et al.
(2005). First, Zn2+was removed from the protein by incubating
overnight in a solution of MBS containing 5 mM Na2EDTA and
2 mM 1,10-phenanthroline. Next, chelators were separated from
protein by gel filtration (BioRad 10DG column) in MBS. The
reductant Tris[2-carboxylethyl] phosphine (Molecular Probes)
was added to 300 mM and incubated for 10 min to reduce the
zinc finger disulfide to thiols. Reduced protein was diluted to
1.5 mM in MBS and pipetted at a volume of 100 mL into each
well of a 96-well Reacti-Bind maleimide-activated clear strip
plate (Pierce) that had been previously rinsed twice with 200 mL
of MBS. The plate was shaken at room temperature in the dark
for 4 h, after which the wells were rinsed four times with 200 mL
Titration with ligand
Fluorophore-labeled proteins that were titrated in solution were
diluted to 10–30 nM in MBS in a fluorescence cuvette with
constant stirring. To this solution was added incrementally
increasing amounts of D-(?) ribose or D-(+) glucose (Sigma)
dissolved in MBS. After each addition of ribose, the fluores-
cence at 666 nm (Cy5) or 570 nm (Cy3) was recorded using
a Tau3 Fluorolog spectrofluorometer (Horiba Jobin Yvon). A
titration curve (see Results) was obtained by fitting the data
(fluorescence as a function of ribose concentration) to a hyper-
bolic binding isotherm for a two-state model (Marvin et al.
where F is the fluorescence at ligand concentration [S], Kdis
the dissociation constant, and FFand FBare the fluorescence
intensities of the ligand-free and ligand-saturated states,
respectively. The signal transduction parameter DFmaxis defined
as the fractional change (FB/FF) ? 1.
Proteins immobilized to microtiter plates were titrated in an
array of wells consisting of two to three rows of eight to 12
wells each. Fluorescence emission from each well in the array
was recorded with a SpectraMax GeminiXS microplate reader
(Molecular Devices). Instrument settings were as follows: PMT
voltage, high; reads per well, 30; and autocalibration, on. The
temperature was regulated at 25°C. Optical parameters for Cy5
were 640 nm excitation, 665 nm cutoff, and 680 nm emission.
For Cy3 these parameters were 530 nm excitation, 550 nm
cutoff, and 570 nm emission. After loading with protein, the
wells were rinsed, usually four times, until the fluorescence
emission remained constant. The wells were filled with 200 mL
of MBS and fluorescence emission recorded two or three times.
In the case of TteRBP, 2 mL of ribose solution was added to each
well. Replicate wells in the array received aliquots of ribose
stock, which ranged from 10 nM to 3 mM in decade intervals
along the rows of the array. In the case of TmGBP, the initial
buffer was decanted from the wells and replaced with 200 mL of
glucose solution. The sugar solutions were mixed, and fluores-
cence was recorded. Two or three recordings of fluorescence
were made after addition of ligand, and the average reading per
well was used for further data processing. For each well, the
ratio of fluorescence in the presence of ligand to fluorescence
without ligand was computed. The average of this ratio was
computed for replicate wells. This average ratio, as a function of
sugar concentration, was fit to the equation above, but fluores-
cence intensity was replaced with fluorescence ratio.
Kinetics of fluorescence recovery
Cy5-labeled TteRBP-CZif, TmGBP-CZif, or TmGBP-NZif was
immobilized to 16 wells (two strips) of maleimide-activated
wells as described above. For TteRBP-CZif, ribose in MBS was
added to all wells at a concentration of 30 mM. For TmGBP-
CZif and TmGBP-NZif, glucose in MBS was added to all wells
to 1 M. Fluorescence emission from the wells was recorded, and
the wells were rinsed four times with MBS to dilute residual
sugar solution to <1 nM. Next 200 mL of MBS was added to
eight wells (one strip), and 200 mL of the original sugar
concentration was added to the remaining eight wells. Within
135 sec of rinsing, the plate was placed in the microplate reader
and the emission at 680 nm was recorded at equal time intervals,
with shaking during the interval to mix the contents of the wells.
For each time point, the fluorescence from the eight wells
containing MBS and from the eight wells containing sugar was
separately averaged. Next, the ratio of fluorescence from MBS
wells to fluorescence from wells containing saturating sugar
concentration was computed for each time point. This ratio was
plotted as a function of time and fit to a single exponential
function to derive a pseudo-first-order rate constant (see
We thank David Goad, Lara Wald, and Tiffany Thoren of
Nomadics, Inc., and Shahir Rizk, James Qiu, and Matt Cuneo
of Duke Univeristy for helpful discussions. Janel Lape and
Gregory Shirman provided assistance with gene construction
and protein purification. This work was funded by grants from
the U.S. Department of Homeland Security (W81XWH-05-C-0161)
and the NIH Director’s Pioneer Award (5 DPI OD000122).
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