Site-Specific Labeling of Surface Proteins on Living Cells Using GeneticallyEncoded Peptides that Bind Fluorescent Nanoparticle Probes

Mark A. Rocco,† Jae-Young Kim,‡ Andrew Burns,§ Jan Kostecki,† Anne Doody,† Ulrich Wiesner,§ andMatthew P. DeLisa*,†,‡

Department of Biomedical Engineering, School of Chemical and Biomolecular Engineering, and Department of MaterialsScience and Engineering, Cornell University, Ithaca, New York 14853. Received January 9, 2009; Revised Manuscript ReceivedJuly 8, 2009

We report a highly specific, robust, and generic method for noncovalent labeling of cellular proteins with highlyfluorescent core-shell silica nanoparticles termed C dots. Our approach uses short genetically engineered peptideswith affinity for silica (GEPS) that are site-specifically introduced at the termini or in loops of cellular proteins.Because GEPS are absent from native cell surface proteins, GEPS-tagged recombinant proteins can be selectivelyand rapidly labeled with fluorescent C dots. To demonstrate the versatility of our method, we targeted 30 nm Cdots to two structurally distinct integral outer membrane proteins in Escherichia coli, FhuA and OmpX. Efficientlabeling was achieved in 15 min or less and was observed to be highly sensitive and specific. This strategyprovides a powerful technique, comparable to other chemical and biological labeling strategies, for efficient andquantitative investigation of protein function in live biological cells.

INTRODUCTION

Visualizing the movement and interactions of cellular proteinsis fundamental to our understanding of biochemical networksand for elucidating the structure and function of proteins in theirnative environment. Numerous biophysical probes (e.g., fluo-rophores, spin labels, etc.) have been developed for labelingcellular proteins with excellent spatiotemporal resolution (1, 2).However, a primary factor limiting the use of many such probesis the technological challenge associated with probe conjugationto target proteins. To address this challenge, a number of generallabeling strategies have been reported recently that employspecial protein or peptide handles for recruitment of biophysicalprobes to specific sites within proteins on the surface of livingcells (3-6). For instance, Ting and co-workers have developeda two-step conjugation procedure based on enzymatic biotiny-lation that enables cell surface protein labeling using differentprobes, including fluorescent semiconductor nanoparticles knownas quantum dots (QDs) (7). QDs hold great promise asbiophysical probes because of their intense fluorescence emis-sion and remarkable photostability (8, 9), as well as their abilityto be rendered water-soluble and thus used in biological mediaand other aqueous environments (10). However, although QDsexhibit many desirable photophysical properties, they face sig-nificant challenges relating to their toxicity and disposal (11, 12).

As an alternative to QDs, organic fluorophores can beincorporated into nanoparticles during synthesis (13-16). Forinstance, Wiesner and co-workers recently developed a classof monodisperse fluorescent core-shell silica nanoparticles,termed C dots (Figure 1a), by integrating covalently bound dyesin a sol-gel-derived silica matrix (17). The particles areassembled in a core-shell architecture via a modified Stobersynthesis (18) with the dye molecules (e.g., tetramethyl-

rhodamine isothiocyanate, TRITC) sequestered within theparticle core, which in turn is enclosed in a layer of pure silica.This architecture results in a variety of improved properties,such as reduced photobleaching, minimized solvatochromicshift, and increased fluorescence efficiency relative to free dyein aqueous solution and provides particles with sizes down tothe 3-5 nm scale (19, 20). The C dot design also allowscolocalization of multiple fluorophores within a single particle.This significantly increases per-particle brightness and, becausethe encapsulated dye molecules are decoupled from each other,reduces intermittent blinking under continuous excitationsalimitation that hinders single-emitter systems such as greenfluorescent protein (GFP) (21) and QDs (22). Importantly, Cdots have found use in a broad range of biological imagingapplications and are non-cytotoxic at biologically relevantconcentrations (17, 23). Given the vast diversity of organic dyemolecules and the exquisite chemical and photophysical tun-ability of different encapsulation matrices, this approach shouldenable the development of nanoparticles with a broad range ofprecisely controlled fluorescence characteristics.

Here, we present a generic method to rapidly and reversiblylabel proteins on the surface of living cells with highlyfluorescent silica nanoparticles. This was accomplished byexploiting short 12-mer peptide sequences, which we term GEPS(genetically engineered peptides for silica), as recognitionelements for site-selective labeling of cell surface proteins with30 nm C dots. Due to the ability of these peptides to bindbiogenic silica (24), we hypothesized that they could be usedto create a noncovalent linkage between the organosilicateexterior matrix of C dots and proteins of interest. Indeed, wedemonstrate that these tags can be genetically encoded intoregions of a target protein sequence, such as at the termini orin loops, where they do not disturb the protein’s structure andfunction but are accessible to exogenously added C dots.

EXPERIMENTAL PROCEDURES

Synthesis of C Dots. To achieve the core-shell architectureused for the genetically encoded fluorescent silica nanoparticles,a modified Stober sol-gel process was used as described

* Address all correspondence to: Matthew P. DeLisa, 254 Olin Hall,Cornell University, Ithaca, NY 14853 USA; phone: 607-254-8560; fax:607-255-9166; e-mail: md255@cornell.edu.

† Department of Biomedical Engineering.‡ School of Chemical and Biomolecular Engineering.§ Department of Materials Science and Engineering.

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10.1021/bc9000118 CCC: $40.75 2009 American Chemical SocietyPublished on Web 08/03/2009

previously (17). Briefly, alkoxysilanes were hydrolyzed andcondensed in an aqueous alcohol solution with ammonia actingas catalyst, in the presence of the organic dye TRITC, covalentlyencapsulating of a silane derivative of the fluorescent dyemolecules into the silica matrix (17, 25, 26). This dye-rich coreformation reaction was followed by a second reaction withfurther pure silica precursor to form the particle shell. Varyingthe reaction chemistry allows specific tailoring of the desirednanoparticle size as described (17).

Peptide Labeling of C Dots. Proof-of-concept experimentswith purified peptides, were performed by incubation of 60 nmC dots with 12-mer oligopeptides (Sigma-Genosys; Ge4-1 -TVASNSGLRPAS and Si4-1 - MSPHPHPRHHHT) conju-gated to Texas Red C2 maleimide (Invitrogen, Inc.) followedby repeated wash steps using water to remove unbound peptide.Following each wash, microcentrifugation was used to pelletthe 60 nm particles, and pelleted particles were resuspended inwater prior to fluorometric analysis.

Bacterial Strains and Plasmids. Escherichia coli strainDH5R (F- φ80dlacZ∆M15 ∆(lacZYA-argF)U169 deoR nupGrecA1 endA1 hsdR17(rk

-, mk+) phoA glnV44 thi-1 gyrA96 relA1

λ-) was used for cloning and labeling experiments. E. coli strainsAB2847 (F- aroB thi tsx malT) and MB97 (as AB2847, but∆fhuA) were used for the albomycin resistance experiments (27).All plasmids generated in this study were derivatives of pHIE11(28) or pBAD24. All FhuA constructs were created by insertingDNA encoding the silica- or germania-binding sequences intothe FseI-NotI multicloning site of plasmid pHIE11 (28). Thisresulted in the placement of each GEPS sequence in extracellularloop 5 of FhuA, just after the proline at position 405. For theOmpX constructs, the E. coli ompX gene was PCR-amplifiedusing genomic DNA as template and appended with a C-terminal 6X-histidine tag. The resulting PCR product was clonedin between the NcoI and SphI sites of plasmid pBAD24 (29)resulting in plasmid pBAD24-OmpX-his. To use extracellularloop 2 in OmpX for the insertion of silica-binding peptides,KpnI and SpeI sites were inserted between the serine residuesat positions 53 and 54 of OmpX by overlap extension PCR.The resulting PCR product was digested with NcoI and SphIand ligated into pBAD24, resulting in pBAD24-OmpX*-his.DNA encoding each of the 12-mer GEPS sequences was clonedin between KpnI and SpeI of pBAD24-OmpX*-his. PlasmidspHIE11 and pBAD24-OmpX*-his were modified to includeeach of the following GEPS primary sequences: Ge4-1 -TVASNSGLRPAS; Si3-3 - APPGHHHWHIHH; Si3-8 -KPSHHHHHTGAN; Si4-8 - APHHHHPHHLSR; and Si4-1 -MSPHPHPRHHHT. All GEPS sequences were codon optimizedfor expression in E. coli. All plasmids were confirmed bysequencing.

Labeling of Living Cells with C Dots. Cells containingFhuA or OmpX plasmids were grown overnight at 37 °C inLuria-Bertani (LB) medium containing appropriate antibiotics(50 µg/mL kanamycin for all FhuA constructs and 100 µg/mLampicillin for all OmpX constructs). The following day,overnight cells were diluted 10-fold in LB containing antibioticsand allowed to grow for 90 min at 37 °C (OD600 ∼0.5).Subcultured cells were induced with 1 mM IPTG for 90 min at37 °C, at which time the cultures were pelleted via centrifugationfor 15 min at 4 °C and 13,200 rpm. Pelleted cells wereresuspended in 1 mL of phosphate buffered saline (PBS) andnormalized to an OD600 of 1.0. Normalized samples wereincubated in the dark for 1 h at 4 °C with 30 nm TRITC C dotsat a concentration of 8.1 × 108 particles/µL, unless otherwisenoted, followed by an additional three washes in PBS.

Fluorescence Microscopy. C dot-labeled cells were imagedusing a Carl Zeiss Axioskop 40 optical microscope with a Zeiss100×/1.30 Oil Plan-NEOFLUAR lens, an EXFO X-Cite Series120 lamp (Mississauga, Ontario), a TRITC emission filter, anda SPOT FLEX digital camera from Diagnostic Instruments, Inc.(Sterling Heights, MI). A total of 5 µL of live bacterial cellswas placed onto a microscope slide and a coverslip placed ontop. A drop of oil was added and images were taken using anoil-immersion technique at a magnification of 1000×.

Fluorescence Microplate and Flow Cytometric Analysis. Cdot-labeled cells were aliquoted into a 96-well microtiter plate(Corning COSTAR) in triplicate and fluorescence was quantifiedusing a fluorescence microplate reader (Bio-Tek Synergy HT).Cells were excited at a wavelength of 530 nm, and total cellfluorescence was measured at an emission wavelength of 590nm, parameters that were near-optimal for TRITC (ex, 557 nm;em, 576 nm). Data were reported as an average of threemeasurements with an error determined as the standard deviationof the mean. For flow cytometric analysis, 5 µL of C dot-labeledcells were diluted into 1 mL of PBS and single-cell fluorescencedistributions were measured using a Becton Dickinson FACS-Calibur flow cytometer as described elsewhere (30). Theexcitation/emission filters for this system were configured at488 and 585 nm, respectively. For TRITC C-dots, a 488 nmexcitation wavelength only excites at about 1/6 of the peakabsorption and collects at about 80% of the peak emission.

Protein Analysis. To analyze protein expression, solublefractions were prepared from 10 mL cultures of E. coli cellsexpressing the different FhuA and OmpX constructs as follows.First, cells were pelleted by centrifugation for 15 min at 4 °Cand 3500 rpm and then resuspended in 1 mL PBS solutionfollowed by sonication (Branson Sonifier). Then, the sonicantwas spun for 15 min at 4 °C and 13,200 rpm, and the resultingsupernatant was collected as the soluble fraction. Proteins in

Figure 1. (a) Scanning electron micrograph of 30-nm-diameter TRITC C dots. This size particle was used for all live cell labeling studies. (b)Schematic of binding between C dot and different peptide sequences. Short, purified peptides were used for proof-of-concept binding experimentsusing orthogonal C dot-peptide pairs. For these experiments, Texas Red conjugated oligopeptides (either silica-binding (Si4-1) or germania-binding (Ge4-1) peptides) were incubated with 60 nm core-shell fluorescent silica nanoparticle C dots that incorporated tetramethylrhodamineisothiocyanate dye (TRITC) as shown.

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the soluble fractions were separated by SDS-PAGE using 12%Tris-HCl gels (BioRad) and Western blotted as described (31).For all pFhuA constructs, anti-FhuA (kindly provided by Dr.Andreas Meinke, InterCell Biomedizinische Forschungs undEntwicklungs AG) was used as the primary antibody at adilution of 1:100 000 in conjunction with a horseradish peroxi-dase-conjugated antirabbit secondary antibody (Sigma) diluted1:5000. For all pOmpX constructs, horseradish peroxidase-conjugated antipolyhistidine antibody (Abcam) was used at adilution of 1:3000.

Albomycin Resistance Assay. Growth inhibition by albomycinwas assayed using a protocol similar to that described by Braunand co-workers (32). Briefly, strain MB97 was transformed withplasmids pFhuA, pFhuA-Ge4-1, or pFhuA-Si4-1. Liquidcultures were grown overnight until stationary phase at 37 °Cin TY media (1.0% tryptone, 0.5% yeast extract, and 0.5%NaCl). 100 µL of each overnight culture was centrifuged andresuspended in fresh TY media to eliminate any cellular wasteand debris. These cells were serial diluted (down to a dilutionof 106) and spotted on TY/agar plates containing no antibioticsor 25 µg/mL albomycin. Plated cells were grown overnight at37 °C and subsequently imaged.

RESULTS

Identification of Orthogonal Peptide-Nanoparticle PairsUsing Purified Peptides. The most common approach fortargeting QDs and C dots to a protein of interest is to conjugatethe nanoparticles with a high-affinity binding protein, such asan immunoglobulin or streptavidin, which mediates binding ofthe nanoparticle to a specific antigen or biotinylated protein,respectively (9, 17). However, the large size of this complexcan interfere with membrane protein trafficking and can reduceaccessibility of the nanoparticles. Therefore, our goal was todevelop a generic method for direct labeling of cell surfaceproteins with C dots that eliminated the need for bulky bindingproteins and provided a genetically encodable linkage betweenthe protein of interest and the C dot. This was accomplishedusing genetically engineered peptides for silica (GEPS), whichare short amino acid sequences that specifically and selectivelybind silica. Previous studies using filamentous phage displayhave isolated genetically engineered peptides that bind to, forexample, silica (24) and titania (33). In the present study, wehypothesized that the silica-binding peptides would electrostati-cally bind to the surface of C dots. The rationale for this wasbased on the notion that GEPS selected for strong binding to aspecific silica surface of given size and morphology can oftenbind to another surface of the same material or to a differentmaterial but having similar structure or physiochemical proper-ties (34).

We first sought to determine whether GEPS sequences thatwere previously isolated for their ability to bind and precipitatebiogenic amorphous silica (24) could similarly bind C dots.Proof-of-concept binding experiments were performed to evalu-ate binding between C dots and different fluorescently labeledpeptides (Figure 1b). Specifically, we used two synthetic 12-mer peptides: one with high affinity for silica (Si4-1, MSPH-PHPRHHHT) and a second, nonspecific control peptide withaffinity for germania (Ge4-1, TVASNSGLRPAS). A GGGCmotif was incorporated at the C-terminus of each peptide inorder to react the thiol of the C-terminal cysteine with TexasRed C2 maleimide. Dye-labeled silica- and germania-bindingoligopeptides were incubated at various concentrations with 60-nm-diameter TRITC core-shell C dots. Fluorescence spectraand binding curves revealed enhanced binding specificity forthe Si4-1 peptides with the silica nanoparticle surface relativeto that of the Ge4-1 peptides or to the free dye controls (Figure2a,b). It should be noted that since silica has its isoelectric point

around pH 2-3, at neutral pH the particles are stabilized byeletrostatic repulsive interactions (19, 20). At very high saltconcentrations (e.g., around 0.1 M aqueous NaCl), theseelectrostatic repulsions are screened and steric repulsive interac-tions must be used to stabilize the particles. Hence, the peptideand cell labeling conditions (i.e., PBS at neutral pH) used hereare expected to disfavor particle aggregation. It should also benoted that we chose 60 nm C dots for the peptide labelingexperiments because of difficulties associated with microcen-trifugation of smaller C dots (e.g., 30 nm). In later experimentsdescribed below, 30 nm C dots were used because their smallersize was desirable for cell labeling purposes, and due to theirspecific capture by cells, microcentrifugation was no longer anissue.

Rapid, Highly Specific Protein Labeling in Living Cells. Wenext tested whether the orthogonal C dot-peptide pairs couldbe used to label cellular proteins. This was achieved byengineering the high- (Si4-1) and low- (Ge4-1) affinity GEPSinto a highly exposed extracellular loop of the E. coli outermembrane protein, FhuA. FhuA is a ferrichrome and phage T5receptor that exposes 11 loops to the extracellular milieu and10 turn regions to the periplasm (35, 36). Previous studiesdemonstrated that extracellular loops 4 and 5 of FhuA are ableto tolerate insertions of up to 250 and 166 amino acids,respectively, while still maintaining functionality (28). Accord-ingly, we introduced the Si4-1 and Ge4-1 sequences just afterthe proline residue at position 405 in the fifth extracellular loopof FhuA (Figure 3a), a site displaying the largest surface areato the extracellular milieu.

To determine the efficacy of this labeling strategy using livecells, the various FhuA constructs were expressed in E. coliand analyzed via fluorescence microscopy. Following labelingwith 30 nm C dots at a final concentration of 8.1 × 108 particles/µL, cells expressing the FhuA-Si4-1 construct showed a highlyfluorescent phenotype, whereas cells expressing FhuA-Ge4-1or wild-type FhuA with no peptide insert exhibited only minor,

Figure 2. Proof-of-concept binding experiments using C dots and short,fluorescently labeled oligopeptides. (a) Fluorescence spectra and (b) bindingcurves for the orthogonal C dot-peptide pairs shown in Figure 1b. TexasRed-labeled silica- and germania-binding 12-mer peptides (Si4-1 andGe4-1, respectively) were incubated with 60 nm TRITC C dot particlesand analyzed via a fluorescence spectrometer. Free TRITC dye was usedas a control. Data represent the average of three replicate experiments.The standard error in each case was less than 5%.

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static background fluorescence (Figure 3b). Background fluo-rescence was readily distinguishable from target fluorescencedue to both the brightness and localization of the C dots.Characteristic nonspecific background fluorescence appeared asvery bright C dot clusters that were not colocalized with E. colicells. In contrast, positive target fluorescence corresponded toa halo-like fluorescent ring that colocalized with the E. coli cellbody (Figure 3b) and was similar to the fluorescence patternseen for other cell surface or periplasmic labeling strategies (37).The same C dot-treated cells were subjected to fluorescence-activated cell sorting (FACS) analysis. Consistent with themicroscopy results, cells expressing FhuA-Si4-1 showed a3-fold increase in mean cell fluorescence (MF) compared to cellsexpressing FhuA-Ge4-1 or wild-type FhuA (Figure 3c). Whenthese same cells were analyzed using a fluorescence microplate

reader, we observed a 5-fold difference in fluorescence betweencells expressing FhuA-Si4-1 and cells expressing FhuA-Ge4-1or wild-type FhuA (Figure 3d). We attribute the improvedsignal-to-noise obtained using the microplate reader to theexcitation/emission filters (ex, 530; em, 590 nm) of this device,which match the excitation/emission wavelengths of TRITC (ex,557 nm; em, 576 nm) more closely than our current flowcytometry setup. Moreover, cells expressing FhuA-Si4-1exhibited a C dot concentration-dependent fluorescence responsefollowing labeling with 2.7 × 108 to 2.7 × 109 particles/µL.This was not the case for cells expressing FhuA-Ge4-1 or wild-type FhuA; each of these resulted in only background fluores-cence following labeling over the same C dot concentrationrange (Figure 3d). At the highest concentration of C dots tested(2.7 × 109 particles/µL), the fluorescence signal for FhuA-Si4-1

Figure 3. (a) Schematic and three-dimensional structural representations of FhuA depicting the GEPS insertion site in extracellular loop 5, immediatelyfollowing a proline residue at position 405 (P405). In the FhuA crystal structure (right), loop 5 is highlighted in blue and the GEPS sequence ishighlighted in red. (b) Fluorescence microscopy of cells expressing FhuA-Si4-1, FhuA-Ge4-1, or wild-type FhuA with no peptide insert. Followinglabeling with 30 nm TRITC C dots at a concentration of 8.1 × 108 particles/µL, 5 µL of live bacterial cells were placed onto a microscope slideand a coverslip placed on top. A drop of oil was added and images were taken using an oil immersion technique at a magnification of 1000×. Insetis an enlarged image of a single cell. (c) FACS analysis of the same constructs described in (b). Shown are representative FACS histograms. Meanfluorescence (M) values are the average of three replicate experiments. The error in each case was less than 5%. (d) Quantification of cell fluorescenceafter C dot labeling. Cells were induced to express FhuA-Si4-1 (gray bars), FhuA-Ge4-1 (white bars), and wild-type FhuA (black bars) and thenlabeled with a range of C dot concentrations as indicated. Data are the average of three replicate experiments, and error bars represent the standarddeviation of the mean. (e) Western blot analysis of soluble fractions generated from cells expressing FhuA-Si4-1, FhuA-Ge4-1, and wild-typeFhuA. Soluble proteins from an equivalent number of cells were loaded in each lane and detection was with FhuA antiserum.

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was ∼9× greater than the FhuA-Ge4-1 control, verifying thespecificity of the GEPS insert. Finally, it is noteworthy that,even at the highest C dot concentration, there was no measurablegrowth inhibition for any of the cells tested (data not shown),confirming the biocompatibility of the silica nanoparticles.

We next determined whether the enhanced cellular fluores-cence seen for FhuA-Si4-1 versus FhuA-Ge4-1 was due tospecificity/affinity of the peptide sequences for the C dots or,instead, differential protein expression. To exclude variationsin protein expression, Western blot analysis was performed onsamples prepared from cells expressing each of the FhuAconstructs. As expected, there was virtually no difference insoluble expression of FhuA-Si4-1 and FhuA-Ge4-1 (Figure3e), suggesting that the differential fluorescence observed abovewas attributable to varying degrees of affinity conferred by thepeptide inserts. To investigate this in more detail, we constructedseveral additional FhuA constructs by inserting other silica-specific peptides of varying affinities into extracellular loop 5

of FhuA. These peptides exhibited intermediate binding of silicathat was less than the high-affinity Si4-1 clone but greater thanthe nonspecific Ge4-1 clone, with the relative binding affinityas follows (in order from highest to lowest): Si4-1 . Si3-3≈ Si3-8 ≈ Si4-8 . Ge4-1 (24). Following C dot labelingof cells, we observed intermediate fluorescence for each of theseFhuA constructs that was reproducibly lower than that for FhuA-Si4-1 but significantly higher than that for the FhuA-Ge4-1construct (Figure 4a). As seen above, similar expression levelswere observed for each of these FhuA variants (Figure 4b)indicating that differences in fluorescence were linked to affinityof the displayed peptides for the C dots. A major advantage ofthis strategy is that high levels of labeling (>80% of themaximum signal) could be achieved in as little as 15 min (Figure4c).

To determine whether labeling was disruptive to proteinfunction, we next evaluated whether the GEPS-modified FhuAvariants retained wild-type activity following labeling with Cdots. Native FhuA in the outer membrane of E. coli activelytransports ferrichrome and the antibiotics albomycin and rifa-mycin CGP 4832. In the case of albomycin, the antibiotic isinternalized through FhuA after which it is cleaved and theantibioticallyactivemoiety is released into thecytoplasm(27,32).Cells expressing functional FhuA are sensitive to albomycin,

Figure 4. (a) Quantification of cell fluorescence following expressionof FhuA constructs modified with different GEPS sequences. Alllabeling was accomplished with 30 nm particles at a final concentrationof 8.1 × 108 particles/mL. Data are the average of three replicateexperiments. (b) Western blot analysis of soluble fractions generatedfrom cells expressing the different FhuA constructs as indicated. Solubleproteins from an equivalent number of cells were loaded in each laneand detection was with FhuA antiserum. (c) Effect of C dot incubationtime on labeling efficiency. Cells expressing FhuA-Si4-1 wereincubated with 30 nm particles at a final concentration of 8.1 × 108

particles/mL for the indicated labeling times. Data are the average ofthree replicate experiments and are normalized to the florescence signalobtained for FhuA-Si4-1 at 60 min, which was set at 100%. All errorbars represent the standard deviation of the mean.

Figure 5. Albomycin resistance phenotype of FhuA-expressing cells.Spot plating of wt AB2847 and isogenic MB97 (∆fhuA) cells on TY/agar plates with (a) no antibiotic or (b) 25 µg/mL albomycin. Plasmid-based complementation of fhuA in MB97 cells was from pFhuA,pFhuA-Si4-1, or pFhuA- Ge4-1, as indicated. Spot plating of thesame cells in (a,b) following labeling with 30 nm TRITC C dots for1 h prior to plating on (c) no antibiotic or (d) 25 µg/mL albomycin.Cells were serially diluted as indicated above each panel. (e) Quanti-fication of cell fluorescence after C dot labeling of AB2847 or MB97cells carrying no plasmids or expressing wild-type FhuA, FhuA-Ge4-1,and FhuA-Si4-1 as indicated. Cells were incubated with 8.1 × 108 Cdots/µL for 1 h. Data are the average of three replicate experimentsand error bars represent the standard deviation of the mean.

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whereas those lacking a functional FhuA protein do not takeup albomycin and are resistant to the antibiotic. To determinewhether the 12-mer peptide insertions disrupted FhuAfunction, we evaluated the ability of each FhuA construct tointernalize albomycin. As anticipated, AB2847 cells thatexpress an intact copy of wild-type fhuA from their genomewere highly sensitive to albomycin (Figure 5a,b). In contrast,E. coli strain MB97, which carries a complete deletion ofthe fhuA gene (27) and thus should not internalize albomycin,grew normally on agar plates supplemented with albomycin.However, when MB97 cells were provided with a plasmid-encoded copy of FhuA, FhuA-Si4-1, or FhuA-Ge4-1, theybecame highly sensitive to albomycin (Figure 5a,b). Thus,we conclude that the insertion of the peptides in FhuA didnot disrupt function because each of the FhuA constructs wascapable of internalizing albomycin. Next, to determinewhether labeling of FhuA with C dots inhibited function,perhaps by cross-linking FhuA into inactive multimers, weperformed a similar experiment as above except now the cellswere labeled with C dots for 1 h prior to plating onalbomycin. The albomycin resistance behavior of C dotlabeled cells was identical to that observed for the same cellsthat had not been treated with C dots (compare Figure 5c,dwith a,b), indicating that C dot labeled FhuA remainedfunctional. It is noteworthy that the fluorescence signal forMB97 cells expressing the FhuA-Si4-1 was nearly 20-foldabove the background signal from FhuA-Ge4-1 expressingcells (Figure 5e), which was an improvement over the signal-to-background ratio observed above using DH5R host cells.

Evidence for a Universal Targeting Strategy. To determinewhether our C dot binding strategy was generic, we introducedthe panel of different GEPS into the E. coli integral outer

membrane protein OmpX. Like FhuA, OmpX is a �-barrelmembrane protein. It contains eight transmembrane �-strandsand exposes four extracellular loops (38). We introduced eachof the different GEPS into extracellular loop 2, just after theserine residue in position 53 (Figure 6a). In agreement with theFhuA results, cells expressing OmpX-Si4-1 were nearly 5 timesmore fluorescent than their OmpX-Ge4-1-expressing counter-parts, as revealed by both FACS and plate reader measurements(Figure 6b,d). Similarly, cells expressing the intermediatepeptide sequences exhibited levels of fluorescence that were lessthan that seen for OmpX-Si4-1 but significantly greater thanOmpX-Ge4-1. It is noteworthy that Si3-3 resulted in thelowest fluorescence of the three intermediate binders wheninserted in FhuA but was the most fluorescent of the three whencloned in OmpX (Figure 6d). It appears, however, that thisunexpectedly high fluorescence was due to a marked increasein OmpX-Si3-3 expression relative to all other OmpX con-structs (Figure 6c). For the other two intermediate binders, therelative C dot binding affinity observed with OmpX was thesame as FhuA, with Si4-8 showing greater affinity than Si3-8.For these constructs, as well as OmpX-Si4-1 and OmpX-Ge4-1, the soluble expression levels were virtually indistin-guishable (Figure 6c), suggesting as above that fluorescencelevels were indicative of specificity/affinity for C dots and notgross differences in protein expression levels. Finally, high levelsof labeling (>80% of the maximum signal) were observed in aslittle as 5 min (Figure 6e).

DISCUSSION

In the present study, we report a highly specific, robust, andrapid new method for labeling cell surface proteins withfluorescent nanoparticles, i.e., core-shell silica nanoparticles

Figure 6. (a) Structural representation of OmpX depicting the GEPS insertion site in extracellular loop 2, immediately after a serine residue atposition 53. Loop 2 is highlighted in blue and the GEPS insertion site is indicated by the arrow. (b) FACS analysis of cells induced to expressOmpX-Ge4-1 (left) and OmpX-Si4-1 (right) following labeling with 30 nm particles at a final concentration of 8.1 × 108 particles/µL. Shownare representative FACS histograms. Mean fluorescence (M) values are the average of three replicate experiments. The error in each case was lessthan 5%. (c) Western blot analysis of soluble fractions generated from cells expressing different OmpX constructs as indicated. Soluble proteinsfrom an equivalent number of cells were loaded in each lane and detection was with antipolyhistidine antibodies. (d) Quantification of cell fluorescenceafter C dot labeling of cells expressing OmpX constructs modified with different GEPS sequences. All labeling was accomplished with 30 nmparticles at a final concentration of 8.1 × 108 particles/µL. Data are the average of three replicate experiments. (e) Effect of C dot incubation timeon labeling efficiency. Cells expressing OmpX-Si4-1 were incubated with 30 nm particles at a final concentration of 8.1 × 108 particles/µL for theindicated labeling times. Data are the average of three replicate experiments and are normalized to the fluorescence signal obtained for FhuA-Si4-1at 60 min, which was set at 100%. All error bars represent the standard deviation of the mean.

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referred to as C dots. Our approach uses genetically engineeredpeptides with affinity for silica (GEPS) as C dot recognitionelements that can be site-specifically introduced into cellularproteins. The silica-binding peptides used here were previouslyisolated for their ability to precipitate silica (24) and are enrichedin histidine residues, whereas the control germania-bindingpeptide lacked histidine entirely. Along similar lines, severalother groups have reported peptide sequences with affinity forsilica and the majority of these appear to be rich in basic andhydroxyl amino acids (24, 39-41) as well as proline residues(40, 42). Hence, we believe that C dot binding is due toelectrostatic interaction, however, whether other mechanismsare at play cannot be ruled out.

The advantage of GEPS is that they are relatively short peptidesequences that can be genetically encoded at the termini or in loopsof proteins without disturbing the protein’s structure, function, andsubcellular localization. We show here that strategic positioningof GEPS into loops of outer membrane proteins allows forelectrostatic capture of 30 nm fluorescent C dots with labeling timesas short as 5 min. Because only background fluorescence wasobserved following C dot labeling of wild-type bacterial cells, weconclude that nonspecific silica-binding activity is largely absentfrom native cell surfaces. Hence, GEPS-tagged cell surface proteinscan be efficiently labeled with fluorescent C dots with signal-to-background ratios of ∼5-20 depending on the target protein, labelconcentration, and host cell used. It should also be noted thatlabeling approaches that use peptides rather than proteins are lessinvasive but typically sacrifice specificity. However, the use ofnoninvasive silica-binding peptides comprising only 12 residuesdid not suffer from a lack of specificity as evidenced by thedifferential binding observed for the different GEPS clones. SinceGEPS tags are genetically encodable, we anticipate that the labelingtechnology described here can be extended to numerous structurallydiverse membrane proteins in prokaryotic and also perhaps ineukaryotic cells. Such flexibility should enable visualization ofprotein dynamics underlying numerous biological processes,especially considering that C dot labeling did not adversely affectprotein function. However, a potential concern is that the relativelylarge 30 nm particles may bind to several genetically modifiedsurface proteins simultaneously in a manner that reduces themobility of the proteins in the membrane and interferes withdynamic analysis. Therefore, smaller C dot particles may benecessary for performing dynamic analysis. To date, the smallest,stable particles with narrow particle size distributions that we haveachieved go down to about 3 nm (19).

Interestingly, these peptides not only promoted C dot binding,but different primary sequences resulted in differential bindingefficiency. This implies that it may be possible to further optimizeGEPS with increased affinity for C dots. For instance, cell surfacedisplayed libraries of random Si4-1 variants could be labeled withC dots and screened using FACS to isolate highly fluorescent clonesthat represent ultrahigh affinity binders. A similar affinity matura-tion strategy was demonstrated recently by Rozinov and Nolanwho used phage display to isolate higher-affinity peptides againstsmall-molecule fluorophores such as Texas Red (43). Our studieshere show that C dot labeled cells can be visualized with a flowcytometer indicating that FACS could be used to efficientlydiscriminate binders from nonbinders. Interestingly, while earlierstudies reported comparable silica binding for each of theseintermediate peptides, our studies show distinct binding differencesfor these peptides. We suspect that this discrepancy is due todifferences in how the peptides were displayed during the bindingassays. In the earlier work of Stone and co-workers (24), peptideswere expressed as unconstrained fusions to the minor coat (pIII)protein of M13 phage, whereas in our studies, the peptides wereconstrained in the extracellular loops of an outer membrane protein.

A particularly attractive feature of this technology is that itmakes possible for the first time a system for genetically taggingcellular proteins with epitopes specific for fluorescent silicananoparticles. Importantly, the 30 nm diameter variety testedhere have already been shown to be 20-30 times brighter on aper particle basis and nearly 20 times more stable than theirconstituent dyes (17, 25, 44), offering a robust new avenue forfluorescent labeling. Furthermore, because C dots are multif-luorophore systems, at any given time there will still be a portionof the dyes emitting, conferring continuous fluorescence emis-sion. From a practical standpoint, this minimizes the intermittentblinking under continuous excitation suffered by single-emittersystems (25). Since many different fluorophores (e.g., TRITC,Oregon Green 488, etc.) and even multiple fluorophores can belocalized within a single silica nanoparticle (25, 44), colors andspectral properties, such as peak wavelength and shape, can bechosen at will from a palette of dyes and efficiently coupled tocellular proteins.

In this study, we focused on the integral outer membraneproteins FhuA and OmpX of E. coli. These and other �-barrelsfound in the outer membranes of prokaryotic and eukaryoticorganisms constitute an important functional class of proteins.Such proteins play important roles in bacterial pathogenesis,regulating molecular transport, and mitochondrial homeostasis(45), and therefore are high-priority targets for structural andfunctional characterization. For instance, E. coli OmpX and itshomologues in the human pathogens Yersinia, Enterobacter,Klebisiella, and Salmonella promote adherence to and entry intohost cells, a key step in the ability of these pathogens toneutralize the host defense system and interfere with the hostimmune system (38, 45). Thus, we anticipate that site-specificC dot labeling of these membrane proteins in living cells willpermit multidimensional time-lapse microscopy and single-molecule analysis of pathogen-host interactions.

ACKNOWLEDGMENT

We thank Dr. Andreas Meinke for plasmid pHIE11 as wellas FhuA antiserum. Funding was provided by an NSF CAREERAward CBET-0449080 (to MPD) and a NYSTAR James D.Watson Award (to MPD). We would also like to thank theCornell Nanobiotechnology Center, an STC Program of the NSFunder Agreement No. ECS-9876771 for funding (to UW) andinfrastructure.

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