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: firstname.lastname@example.org; 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
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