Single Molecule Take-and-Place Technique for Positioning a

Article pubs.acs.org/JPCC

Single Molecule Take-and-Place Technique for Positioning a Membrane Protein on a Lipid Bilayer Tseng-Huang Liu,†,§ Yun-Tzu Huang,†,§ Hui-Wen Cheng,⊥ Yen-Wei Chen,† Ching-Hung Lee,† Yu-Di Hsu,† Rong-Long Pan,*,† and Fan-Gang Tseng*,‡ †

Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, ‡Department of Engineering and System Science, and ⊥Center for Nano Science & Technology, National Tsing Hua University, Hsin Chu 30013, Taiwan, ROC

Downloaded by UNIV OF NEBRASKA-LINCOLN on September 3, 2015 | http://pubs.acs.org Publication Date (Web): September 1, 2015 | doi: 10.1021/acs.jpcc.5b05944

S Supporting Information *

ABSTRACT: Manipulating a single protein molecule on liposomes or planar lipid bilayers is a useful technique for studying membrane-bound proteins, receptors, or ion transporters and for delicate applications including biosensor chips, drug screening, and clinical diagnoses. However, several key breakthroughs are required for success through difficult techniques such as single protein take-and-place with reasonable spatiotemporal control. In this report, a novel method was established for placing a single transmembrane protein onto a patterned lipid bilayer. A surface-functionalized 1.8 nm gold nanoparticle was first engineered at the tip of an atomic force microscope. A single transmembrane protein, H+-translocating pyrophosphatase with six-histidine residues tag, was then picked up using such a microscopy tip where its nanoparticle was modified by nickel−nitrilotriacetic acid. Two-step fluorescence bleaching observation and quantum dot blinking analysis subsequently verified successful manipulation of a single functional protein on a lipid membrane in a predetermined manner. Furthermore, the enzymatic activity from the single H+-translocating pyrophosphatase was determined, demonstrating that membrane proteins retain their functions on the lipid bilayer through a single-molecule take-and-place technique. This innovative technique overcomes current limitations and provides a single biomolecule nanomanipulation system for versatile studies of membrane-bound proteins.

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INTRODUCTION Maneuvering a single functional membrane protein onto an artificial liposome or lipid bilayer is an area of intense research and can conceivably be achieved by employing sophisticated single-molecule techniques.1−3 Single-molecule techniques for biological application utilizing optical microscopy, atomic force microscopy (AFM), and optical/magnetic tweezers provide exceptional advantages over conventional methods.4−7 The techniques provide valuable information on fundamental biological processes, such as complicated motions, biochemical reactions, and detailed kinetics, by directly observing and measuring individual biomolecules instead of collecting average phenomenon over a large amount of events.8,9 Furthermore, single molecule techniques may be used to control biomolecules in situ for versatile purposes.10 Manipulations of single molecule membrane proteins also generate new insights on the molecular structures and mechanisms.1 Biomembranes play an indispensable role in maintaining appropriate intra- and extracellular physiological conditions to ensure proper cell function.11−13 Membrane protein function requires a lipid membrane environment that can be also created by reconstituting the protein on artificial liposomes or lipid bilayers.14 Phospholipid bilayer nanodisks have been recently developed as platforms to embed various membrane proteins in a native-like phospholipid bilayer environment.2,3 In addition, © XXXX American Chemical Society

lipid bilayer microarrays/nanoarrays and biosensor systems integrating a variety of membrane proteins can be applied for pharmaceutical screening and investigation of ligand−receptor interactions and ion channel activities.1,15 Further, an array of single membrane proteins has led to remarkable insights into stochastic sensing.1 Nevertheless, reconstituting a single functional membrane protein on a patterned lipid bilayer still presents a challenge because of difficulties in manipulating a single protein molecule with reasonable spatiotemporal control. In this paper, a novel single molecule take-and-place (smTAP) technique was first developed for placing a single functional membrane protein on a patterned lipid bilayer (Figure 1). The smTAP employs a technique used to prepare an AFM probe modified with a single gold nanoparticle (GNP) (Figure 1a,b). The surface of the GNP was further chemically modified for picking up and releasing the protein molecule of interest (Figure 1c, d). Lipid bilayer patches were then formed for protein reconstitution later. A single protein was then transferred and placed on a lipid bilayer patch by AFM tip indentation (Figure 1e,f). After transferring one protein in this Received: June 22, 2015 Revised: August 26, 2015

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DOI: 10.1021/acs.jpcc.5b05944 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

CtH+-PPase were transformed into Escherichia coli strain C43(DE3) (Lucigen, Middleton, WI). The transformants were incubated in Luria−Bertani medium with 50 μg/mL of ampicillin at 37 °C for 2.5 h (until A660 = 0.8). Afterward, 700 μM isopropyl μ-D-thiogalactoside was added, and cultures were further grown for 5 h at 37 °C. The Escherichia coli cells were then harvested by centrifugation at 4000g for 15 min. The Escherichia coli microsomes were isolated according to the procedure described previously.19 The C80A mutant CtH+PPase was solubilized from microsomes by 0.25% (w/v) ndodecyl-β-D-maltopyranoside in an extraction buffer [25 mM MOPS/KOH (pH 7.2), 20% (w/v) glycerol, 400 mM KCl, 4 mM MgCl2, and 1 mM phenylmethanesulfonyl fluoride]. The solubilized CtH+-PPase solution subsequently flowed through the column with nickel−nitrilotriacetic acid beads (Qiagen, Valencia, CA). After washing with 10, 50, and 60 mM imidazole in wash buffer [25 mM MOPS/KOH (pH 7.2), 20% (w/v) glycerol, 400 mM KCl, 4 mM MgCl2, 1 mM phenylmethanesulfonyl fluoride, and 0.05% (w/v) n-dodecyl-β-Dmaltopyranoside], the purified C80A mutant CtH+-PPase was eluted by 250 mM imidazole in elution buffer [25 mM MOPS/ KOH (pH 7.2), 20% (w/v) glycerol, and 0.05% (w/v) ndodecyl-β-D-maltopyranoside]. The purified dimer CtH+PPases were subsequently incubated with cysteine reactive Alexa Fluor 488 and QD 525, individually, according to the standard labeling protocols provided by manufacturer. After 1 h of incubation at 4 °C, unlabeled Alexa Fluor 488 and QD 525 were removed by 30 and 100 kDa cutoff filters, respectively (Millipore, Bedford, MA). All buffer solutions using for fluorophore labeling were degassed and passed through 0.22 μm filter before use. Lipid Bilayer and Stamp Preparation. Small unilamellar vesicles (SUVs) were prepared from soybean phosphatidylcholine (L-α-phosphatidylcholine) with 20% (w/w) cholesterol and DiD dye (10−3 M). The lipid mixture in in chloroform was dried in the vacuum chamber and then suspended in 1 mL of suspension medium [0.25 M sorbitol, 1 mM MgSO4, 0.1 mM EGTA, 2 mM DTT, and 10 mM Tricine-Na (pH 7.5)]. The suspension was sonicated in a bath-type sonicator at 4 °C. Sonicated lipid mixture was then frozen in liquid nitrogen, thawed on ice, and subsequently sonicated for 20 s at 4 °C in a bath-type sonicator. The small unilamellar vesicles thus obtained were stored at 4 °C in the dark. Supported planar lipid bilayers were then prepared by vesicle fusion to the glass coverslip for lipid bilayer patches construction. Excess vesicles were gently washed in suspension medium. The polydimethylsiloxane (PDMS) stamp was manufactured by the incorporation of SU8 lithography and PDMS molding. The O2 plasma treatment was used to transform PDMS stamps to hydrophilic.18 Fluorescence Spectroscopy and Single-Molecule Imaging. Single-molecule fluorescence was generated by TIRF excitation on Olympus IX71 inverted microscopy (Olympus, Tokyo, Japan) with an Olympus 60 × /1.45NA objective. The fluorescence signals from Alexa Fluor 488 and QD 525 were generated using an argon laser (488 nm, 10 milliwatts) (Olmpus, Tokyo, Japan) and images from lipid bilayer, DilC18(5), by a diode laser (638 nm, 35 milliwatts), respectively (Omicron Laserage, Rodgau, Germany). The emitted fluorescence was then collected through band-pass filters appropriate to the fluorophores (520-DF-40 for Alexa Fluor 488 and QD 525; 670-DF-40 for lipid bilayer) (Omega Optical, Brattleboro VT). The images were then recorded by a

Figure 1. Working scheme for the single-molecule take-and-place technique. (a, b) Adsorption of a single GNP (orange) at the AFM tip using a bias voltage generated between the nanoparticle and the tip. (c) Functionalization of single GNP-modified AFM probe with nickelnitrilotriacetic acid. (d) Immobilization of a single His6-tagged membrane protein (green) on the AFM tip via interaction with nickel−nitrilotriacetic acid. (e, f) Transfer of a single protein molecule to a lipid bilayer patch. After transferring one protein in this way, the tip is free again for the next take-and-place cycle.

way, the tip is again free to take and place another single protein (Figure 1).

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EXPERIMENTAL METHODS Materials. 16-Mercaptohexadecanoid acid, 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide, sulfo-N-hydroxysuccinimide, L-α-phosphatidylcholine, and cholesterol were purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488 (Maleimide-reactive probe), QD 525 (Qdot 525), and lipophilic carbocyanine fluorescent dye (1,1′-dioctadecyl3,3,3′,3′-tetramethylindodicarbocyanine perchlorate; DilC18(5), “DiD”) were obtained from Invitrogen (Carlsbad, CA). GNP (1.8 nm) and N,N-bis(carboxymethyl)-L-lysine hydrate were purchased from Nanoprobes (Yaphank, NY) and Fluka (Milwaukee, WI), respectively. The phosphate fluorescence assay kit was purchased from Biovision (Milpitas, CA). The smTAP technique was performed by NanoWizard AFM (JPK Instruments, Berlin, Germany). Single GNP-Modified AFM probe. The single GNPmodified AFM probe was fabricated by applying a bias voltage (2 V) between the tip (PPP-NCH; Nanosensors, Neuchâtel, Switzerland) and the GNP for 40 ns. Individual GNP-modified AFM probes were subsequently immersed in 1 mM 16mercaptohexadecanoid acid and then activated by 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide before reaction with 5 mM sulfo-N-hydroxysuccinimide. The reactive probe was incubated in 100 mM N,N-bis(carboxymethyl)-L-lysine hydrate and then in 40 mM NiSO4 to form the nickel−nitrilotriacetic acid complex.16 CtH+-PPase Protein Expression and Purification. The CtH+-PPase gene was inserted into pET23d vector (Novagen, Nottingham, UK) with a C terminal His6 tag. Cys80 was then displaced by Ala residue by a QuikChange site-directed mutagenesis method.17 DNA constructs of C80A mutant B


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H+-PPase is a proton-pumping protein that utilizes a metabolic byproduct, inorganic pyrophosphate, as an energy source to generate proton gradient across biological membrane for the secondary transport of ions and metabolites.20 This unique proton pump is homodimeric composed of two identical polypeptides with a molecular mass ranging from 60 to 80 kDa21,22 In this investigation, Clostridium tetani E88 H+PPase (CtH+-PPase) with a His6-tag at the C terminal domain was employed as a model for developing an smTAP technique. The secondary structure of CtH+-PPase shows 16 transmembrane domains across cytoplasmic membrane, eight loops toward cytosolic side, and seven loops along with N and C terminal domains exposing outside the membrane.23 In addition, there are two endogenous cysteine residues in CtH+PPase where Cys80 residue locates on the same side as N and C terminal domains while Cys542 is toward opposite side to them.24 To verify that only a single homodimeric CtH+-PPase protein molecule was absorbed on the AFM tip, C80A CtH+PPase mutant with a mutation at Cys80 leaving the sole cysteine at position 542 was constructed for fluorescence probes labeling. The C80A mutant was then heterologously expressed in Escherichia coli strain C43 (DE3) under control of the T7/lac promoter. The microsome and purified C80A CtH+PPase protein, consecutively prepared as described in the Materials section, were observed on SDS-PAGE and also confirmed by immunoblot analysis using anti-His6 tag antibody (data not shown). The C80A mutant brought an increase (20− 30%) in PPi hydrolysis activity and similar proton translocation activity as compared with wild type (data not shown). This system is thus a suitable model to verify the feasibility of this protocol. Afterward, the Alexa Fluor 488 C5-maleimide fluorophores were covalently labeled to the sole cysteine at position 542 in each subunit of the homodimeric C80A CtH+-PPase. The Alexa Fluor 488 labeled C80A CtH+-PPases were immobilized on the L-polylysine coated coverslip. The fluorescence image from each C80A CtH+-PPase molecule was recorded by total internal reflection fluorescence microscopy (TIRFM) combining an electron multiplying charge coupled device using argon laser (488 nm, 10 mW) excitation (Figure 3b, top). For estimating the statistical accuracy in single molecule detection, high rate images were simultaneously recorded to depict the time traces of stepwise fluorescence photobleaching.24,25 As shown in Figure 3b, the highest fluorescence intensity level resulting from two Alexa Fluor 488 fluorophore molecules was initially measured upon illuminating by the 488 nm laser beam. A discrete lapse was soon observed in the intensity of approximate 60% which theoretically resulted from photobleaching on one of the Alexa Fluor 488 fluorophores (Figure 3b, black arrow). Finally, the second drop fallen to background level occurred as the second fluorophore photobleaching (Figure 3b, black arrow). Therefore, the two-step photolysis of each fluorescent spot indicates that individual singlemolecule homodimeric C80A CtH+-PPase conjugated with two Alexa Fluor 488 fluorophores was successfully determined in the current fluorescence image detection system.21,24,25 Accordingly, it is conceivable the AFM tip modified with nickel nitrilotriacetic acid GNPs (see above) was employed to capture a single His6 tagged fluorescently labeled CtH+-PPase (Figure 3c, left). A fluorescence signal corresponding to the emission of Alexa Fluor 488 (at 520 nm) from this single dually labeled C80A CtH+-PPase molecule at the AFM tip was observed as well by TIRFM with 488 nm laser excitation (Figure 3c,

Sensicam electron-multiplying charge coupled device camera (Cook, Romulus MI) and analyzed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).

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RESULTS AND DISCUSSION For smTAP, a GNP as small as 1.8 nm in diameter was attached to the AFM tip by applying a suitable current limited bias

Figure 2. AFM probe modified with a single GNP. (Top) Schematic diagrams of the preparation of an AFM probe harbored at a single GNP. (Bottom) AFM images of GNPs on the matrix before and after applying voltage. The broken-line circles indicate where a GNP was picked up.

voltage between the GNP and the AFM tip; the attachment was monitored by AFM.16 Initially, GNPs were randomly spread on a doped silicon substrate and imaged by AFM in the tapping mode (Figure 2; left). The AFM tip was then placed directly in contact with a selected GNP by employing the force spectroscopy mode. Upon application of bias voltage between the selected nanoparticle and the tip, the nanoparticle was adsorbed onto the AFM tip, as illustrated in Figure 2, right. This technique offers the following advantages over most other methods: (1) commercial AFM probes can be used directly without pretreatment; (2) an on-demand sub-10 nm nanoparticle can be precisely selected and deposited on the apex of the AFM probe without tedious chemical processes; and (3) the entire process takes less than 5 min. For picking up a single membrane protein molecule, the surface of the GNP attached to the AFM tip was chemically modified (Figure 3a).19 The immobilized GNP was first incubated with an alkanethiol (16-mercaptohexadecanoid acid). Subsequently, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide and sulfo-N-hydroxysuccinimide were used to activate the carboxyl group of the alkanethiols to form a covalent bond with the amine group of N,N-bis(carboxymethyl)-L-lysine hydrate (Figure 3a). After complex formation with nickel, a recombinant membrane protein, proton translocating inorganic pyrophosphatase (H+-PPase; EC 3.6.1.1), which was engineered with a stretch of six consecutive histidine residues tag (His6 tag) at the C terminus, was attached by affinity to the nickel−nitrilotriacetic acid moiety. This approach offers the important advantage that the attached protein remains fully functional and can be oriented via the C or N terminal His6 tag. C


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Figure 3. Detection of a single Alexa Fluor 488-labeled CtH+-PPase molecule at the AFM tip. (a) Scheme of the surface modification to functionalize the single GNP-modified AFM tip by nickel-nitrilotriacetic acid. (b) (Top) Images of single Alexa Fluor 488-labeled C80A CtH+-PPase molecules immobilized on the coverslip using TIRFM with 488 nm laser. (Bottom) Time course of fluorescence intensity for two closely spaced Alexa Fluor 488 molecules, showing two-step photobleaching. (c) (Left) Schematic diagram of a Alexa Fluor 488-labeled CtH+-PPase immobilized at the AFM tip. (Middle) Single molecule fluorescence spot at the AFM tip upon TIRF excitation, as indicated by the arrow and enlarged in the inset. Scale bars: 10 μm. (Right) Time course of fluorescence intensity for single-molecule fluorescence at the AFM tip showing two-step photobleaching.

single molecule C80A CtH+-PPase with a QD 525 was successfully observed in the present single-molecule system.27 Similarly, a GNP modified AFM tip was utilized to capture a single QD 525-labeled CtH+-PPase (Figure 4b, top), and subsequently, a distinct fluorescent spot at the tip was clearly monitored with TIRFM (Figure 4b, top). However, the time trace of fluorescence intensity did not reveal any fluorescent “off” state (blinking suppression) but emitted nearly continuous intensity fluctuations under consecutive illumination (Figure 4b, bottom). In addition, an increase in fluorescence intensity was determined as well (Figure 4b, bottom). Previously, investigation on QD fluorescence clearly demonstrated that the blinking behavior and emission properties of single QD are sometimes interfered with by the surrounding environment.28,29 Materials such as oligo ligands and metal particles suppress the fluorescent “off” states on blinking.28,29 Therefore, the photoblinking suppression in this experiment might be caused by the single GNP presenting at the tip. Taken together, this novel technique was practical to immobilize a single molecule CtH+-PPase on the GNP modified AFM tip.25,30 To construct a lipid membrane environment for transmembrane proteins, the lipid bilayer patch was prepared on a coverslip using a PDMS stamp by a procedure described previously with minor modifications (Figure 5a).18 Figure 5b reveals an optical image of PDMS stamp array patterns. After printing on the supported planar lipid bilayer square, the lipid bilayer can be patterned onto the PDMS stamp (Figure 5c). The lipid bilayer on the PDMS stamp was then transferred to a

middle). Moreover, two-step photolysis from the two fluorophore molecules was also determined (Figure 3 c, right, black arrow). The fluorophore-labeling efficiency for the C80A mutant was determined as 97.7% according to the standard labeling/detection protocols provided by manufacturer (Molecular Probes, Invitrogen). The possibility of other nonspecifically bound protein molecules at the tip could be eliminated. These results are sufficient to demonstrate that only a single fluorescently labeled CtH+-PPase molecule was anchored at the AFM tip.24,25 Similarly, maleimide-functionalized quantum dot 525 (QD 525) was utilized to linked with CtH+-PPase homodimer for estimating the statistical accuracy in the single-molecule detection. Maleimide-functionalized QD 525 with a size of ∼4 nm were prepared from commercially available aminopoly(ethylene glycol) functionalized to cross-react with sulfo Nsuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. According to the predicted three-dimensional structure of CtH+-PPase, the proximity between two Cys542 residues is ∼4 nm speculating that only a QD 525 could anchor to CtH+PPase homodimer.23,26 The fluorescence image of individual QD 525-labeled C80A CtH+-PPase molecules immobilized on the L-polylysine modified coverslip was observed by TIRFM using a 488 nm laser beam (Figure 4a, top). The time traces of the fluorescence intensity displayed fluorescent “on” state and a nonfluorescent “off” state (Figure 4a, bottom). This typical fluorescence blinking phenomenon (on/off events) of QD detected from a fluorescent spot clearly demonstrated that D


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new coverslip forming a patch array (Figure 5d). The epifluorescence image (Emission at 660 nm) of this lipid bilayer circular patch array with DiD dye was then visualized using TIRFM upon excitation by a diode laser (638 nm, 35 milliwatt) (Figure 5d). In addition, the fluorescence signals dropped to the level as background in lipid bilayer square indicating that lipid bilayer was transferred from lipid bilayer square to PDMS stamp (Figure 5c). Ultimately, the AFM tip with the attached single molecule QD 525-CtH+-PPase was then directly inserted on the lipid bilayer patch by NanoWizard AFM. Ethylenediaminetetraacetic acid was subsequently used to remove the nickel ion from the AFM tip. After retraction of the tip from the surface, a single CtH+-PPase protein molecule was reconstituted onto the lipid bilayer. The AFM tip was then free for another take-and-place cycle. The fluorescent spot caused by 488 nm excitation in the evanescent field on the coverslip surface verified the presence of a single QD 525-CtH+-PPase molecule (Figure 6a, left) on the lipid bilayer patch (Figure 6a, right), and the lack of fluorescence on the AFM tip was consistent with the deposition of the QD 525-CtH+-PPase. Furthermore, the on/off photoblinking behavior of QD fluorescence reoccurred following the absence of GNP (Figure 6b). The fluorescence signals switched suddenly between “on” and “off” or “dull” states under continuous excitation with 488 nm laser, again demonstrating the existence of a single QD 525-CtH+-PPase on the lipid bilayer patch. In addition, H+-PPase in the lipid bilayer has been imaged by AFM previously.31 Upon scanning many times by AFM, the protein still remained in the original location of the lipid bilayer.31 This result demonstrated that inserted H+-PPase protein is relatively stationary in the membrane. Moreover, solubilized membrane proteins including CtH+-PPase destabilized by n-dodecyl-β-D-maltopyranoside could be directly incorporated in the performed planar lipid bilayer after removal of nickel ion by ethylenediaminetetraacetic acid.32 We are thus convinced that the solubilized CtH+-PPase proteins were directly inserted into the supported lipid bilayer. Furthermore, QD is too hydrophilic to pass through a lipid bilayer, so QD and cytosolic loops accordingly protruded the lipid bilayer. An AFM image of CtH+-PPase proteins with the QD attached is shown in Figure S1 to confirm that the QDs are always above the lipid bilayer. Herein, the height of almost all of the protrusions on the lipid bilayer is approximately 9 nm (Supporting Information). Therefore, the result confidently validated the feasibility of this system for manipulating a single protein molecule on a lipid bilayer. Subsequently, the enzymatic activity of a single CtH+-PPase molecule in the lipid bilayer patch was assayed using a commercially available Phosphate Fluorescence Assay kit. The reaction solution from the kit was included, and inorganic pyrophosphate hydrolyzed by CtH+-PPase was determined by the progressive appearance of fluorescence detected by an electron-multiplying charge-coupled device. The time course shown in Figure 6c revealed the release of inorganic phosphate, demonstrating that enzyme activity was retained throughout the many steps of smTAP.

Figure 4. Detection of a single QD 525-labeled CtH+-PPase molecule at the AFM tip. (a) (Top) Images of QD 525-labeled CtH+-PPase immobilized on the coverslip using TIRFM with 488 nm laser. (Bottom) Time course of fluorescence intensity for a single QD 525, displaying typical photoblinking. (b) (Upper left) Schematic diagram of a QD-labeled CtH+-PPase immobilized at the AFM tip. (Upper right) Single molecule fluorescence at the AFM tip upon TIRF excitation, as indicated by the arrow and enlarged in the inset. Scale bars: 10 μm. (Bottom) Time course of fluorescence intensity for single molecule fluorescence at the AFM tip.

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CONCLUSIONS This novel mothod, smTAP, employs an AFM tip harboring a surface-functionalized 1.8 nm GNP to successfully transfer individual protein molecules. smTAP allows the reconstitution of a single-membrane protein molecule on a patterned lipid E


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Figure 5. Construction of lipid bilayer patches by PDMS stamp. (a) Schematic illustration of the stamping process by PDMS stamp. (b) Optical images of the PDMS stamp patterns. (c) Epifluorescence images of lipid bilayer square after blotting with a PDMS stamp. (d) Epifluorescence images of a circular lipid bilayer pattern formed by stamping. Scale bars: 70 μm. (2) Nath, A.; Grinkova, Y. V.; Sligar, S. G.; Atkins, W. M. Ligand Binding to Cytochrome P450 3A4 in Phospholipid Bilayer Nanodiscs: the Effect of Model Membranes. J. Biol. Chem. 2007, 282, 28309− 28320. (3) Alami, M.; Dalal, K.; Lelj-Garolla, B.; Sligar, S. G.; Duong, F. Nanodiscs Unravel the Interaction between the SecYEG Channel and Its Cytosolic Partner SecA. EMBO J. 2007, 26, 1995−2004. (4) Müller, D. J.; Dufrêne, Y. F. Atomic Force Microscopy as a Multifunctional Molecular Toolbox in Nanobiotechnology. Nat. Nanotechnol. 2008, 3, 261−269. (5) Zlatanova, J.; van Holde, K. Single-Molecule Biology: What Is It and How Does It Work? Mol. Cell 2006, 24, 317−329. (6) Walter, N. G.; Huang, C. Y.; Manzo, A. J.; Sobhy, M. A. Do-ItYourself Guide: How to Use the Modern Single-Molecule Toolkit. Nat. Methods 2008, 5, 475−489. (7) Jarvius, J.; Melin, J.; Göransson, J.; Stenberg, J.; Fredriksson, S.; Gonzalez-Rey, C.; Bertilsson, S.; Nilsson, M. Digital Quantification Using Amplified Single-Molecule Detection. Nat. Methods 2006, 3, 725−727. (8) Normanno, D.; Vanzi, F.; Pavone, F. S. Single-Molecule Manipulation Reveals Supercoiling-Dependent Modulation of Lac Repressor-Mediated DNA Looping. Nucleic Acids Res. 2008, 36, 2505− 2513. (9) Zhuang, X.; Bartley, L. E.; Babcock, H. P.; Russell, R.; Ha, T.; Herschlag, D.; Chu, S. A Single Molecule Study of RNA Catalysis and Folding. Science 2000, 288, 2048−2051. (10) Neuman, K. C.; Nagy, A. Single-Molecule Force Spectroscopy: Optical Tweezers, Magnetic Tweezers and Atomic Force Microscopy. Nat. Methods 2008, 5, 491−505. (11) Saftig, P.; Klumperman, J. Lysosome Biogenesis and Lysosomal Membrane Proteins: Trafficking Meets Function. Nat. Rev. Mol. Cell Biol. 2009, 10, 623−635. (12) Cho, W.; Stahelin, R. V. Membrane-Protein Interactions in Cell Signaling and Membrane Trafficking. Annu. Rev. Biophys. Biomol. Struct. 2005, 34, 119−151. (13) Yellen, G. The Voltage-Gated Potassium Channels and Their Relatives. Nature 2002, 419, 35−42. (14) Morera, F. J.; Vargas, G.; González, C.; Rosenmann, E.; Latorre, R. Ion-Channel Reconstitution. Methods Mol. Biol. 2007, 400, 571− 585. (15) Cooper, M. A. Optical Biosensors in Drug Discovery. Nature Rev. Drug Discovery 2002, 1, 515−528.

bilayer with reasonable spatiotemporal control. Furthermore, the membrane protein maneuvered retains its enzymatic activity on the lipid bilayer patch. Therefore, smTAP is potentially useful to manipulate various membrane-bound proteins, receptors, or ion transporters in membranes for studying single protein−protein interactions, biochemical reactions, and single-molecule detection.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.5b05944. AFM image of CtH+-PPase proteins with the QD attached in lipid bilayer (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions §

F.-G.T. and R.-L.P. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Science Council, Republic of China (NSC 101-2627-M-007-008, NSC 100-2627-M-007-012, and 100-2311-B-007-001-MY3 to R.L.P.; NSC 101-2627-M-007-003 and NSC 100-2120-M-007-006 to F.G.T.). We thank Dr. Gerhard Gottschalk (Institute of Microbiology and Genetics, Georg-August University, Germany) for his generous gift of genomic DNA of Clostridium tetani E88.

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Figure 6. Reconstitution of a single protein molecule on a lipid bilayer. (a) (Left) Fluorescence image of a single QD 525-CtH+-PPase molecule on a lipid bilayer, as indicated by the arrow and enlarged in the inset. (Right) Fluorescence micrograph of the lipid bilayer patch. (b) Fluorescence intensity trace of QD 525 photoblinking. (c) Time course of the enzymatic reaction of a single CtH+-PPase molecule in the absence or presence of the substrate, PPi. Scale bars: 25 μm. (16) Cheng, H. W.; Chang, Y. C.; Yuan, C. T.; Tang, S. N.; Chang, C. S.; Tang, J.; Chen, F. R.; Pan, R. L.; Tseng, F. G. Simple and Fast Method to Fabricate Single-Nanoparticle-Terminated Atomic Force Microscope Tips. J. Phys. Chem. C 2013, 117, 13239−13246. (17) Kirsch, R. D.; Joly, E. S. An Improved PCR Mutagenesis Strategy for Two-Site Mutagenesis or Sequence Swapping Between Related Genes. Nucleic Acids Res. 1998, 26, 1848−1850. (18) Hovis, J. S.; Boxer, S. G. Patterning Barriers to Lateral Diffusion in Supported Lipid Bilayer Membranes by Blotting and Stamping. Langmuir 2000, 16, 894−897. (19) Zimmermann, J. L.; Nicolaus, T.; Neuert, G.; Blank, K. ThiolBased, Site-Specific and Covalent Immobilization of Biomolecules for Single-Molecule Experiments. Nat. Protoc. 2010, 5, 975−985. G


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