Protocol AFM-Based Guided by Recognition Imaging: A New Mode for Mapping and Studying Interaction Sites at Low Lateral Density

Melanie Koehler 1,2 , Anny Fis 1, Hermann J. Gruber 1 and Peter Hinterdorfer 1,*

1 Institute of , Johannes Kepler University, 4020 Linz, Austria; [email protected] (M.K.); anny.fi[email protected] (A.F.); [email protected] (H.J.G.) 2 Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, 1348 Louvain-La-Neuve, Belgium * Correspondence: [email protected]; Tel.: +43-732-2468-7631

 Received: 19 October 2018; Accepted: 3 January 2019; Published: 8 January 2019 

Abstract: Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction between cognate receptor–ligand pairs leads to new insights into the molecular recognition involved in these processes. Here we present a detailed protocol about applying a technique, which combines (AFM)-based recognition imaging and force spectroscopy for studying the interaction between (membrane) receptors and ligands on the single level. This method allows for the selection of a single receptor molecule reconstituted into a supported lipid membrane at low density, with the subsequent quantification of the receptor–ligand unbinding force. Based on AFM tapping mode, a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor are selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control. The combined mode allows for dynamic force probing on different pre-selected molecules. This strategy results in higher throughput when compared with force mapping. Applied to two different receptor–ligand pairs, we validated the presented new mode.

Keywords: atomic force microscopy; single molecule force spectroscopy; recognition imaging; membrane; receptor–ligand interaction; landscape

1. Introduction Atomic force microscopy (AFM) is one of the most versatile microscopic tools due to its capability to provide not only topographical images, but also to probe and characterize interactions on the single molecular level [1]. Information on unbinding forces, kinetic rate constants, bond life times, and energy landscapes can be obtained. The technique of AFM-based single molecule force spectroscopy (SMFS) is based on the following principle: A functionalized AFM cantilever is approached towards and subsequently retracted from the sample surface at constant velocity in the z-direction at fixed x–y coordinates. The cantilever’s deflection is continuously monitored to record the interaction force, which results in a so-called force-distance cycle (FDC). High densities and homogeneous distributions of receptor molecules are required for performing a successful force spectroscopy experiment, as the tip is “blindly” moved in the z-direction. Such conditions are not always easily applicable, in particular when the molecules of interest are membrane . Only low reconstitution efficiencies are

Methods and Protoc. 2019, 2, 6; doi:10.3390/mps2010006 www.mdpi.com/journal/mps Methods and Protoc. 2019, 2, 6 2 of 18 often obtained for integration into an artificial lipid membrane. To overcome this issue, an AFM-technique known as “force volume (FV)” was initially proposed by Cleveland et al. [2] and further used in pioneering studies by Heinz and Hoh [3] and Gaub et al. [4]. In this mode, the cantilever records one or more FDCs at every lateral position, as it moves across the surface. The main drawback of this handy technique is the acquisition time. The required time for obtaining such a map is directly linked to the selected parameters (image size, pixel resolution, number of FDCs per pixel, FDC sweep time, etc.) and can range from minutes to several hours, when a high-resolution map is needed. Based on the principle of FV, a new mode was developed aiming to overcome the above-mentioned time limitations, i.e., peak force tapping (PFT). Despite the fact that PFT was successfully applied on a variety of biological samples, ranging from membrane proteins [5] to entire cells [6], for high-resolution maps, a few hours are still required. In 2017, a combination of two techniques, simultaneous topographical and recognition (TREC) imaging and SMFS was suggested by Koehler et al. [7]. TREC and SMFS are two AFM techniques that ideally complement each other. Topographical and recognition imaging is an imaging technique that provides information on receptor binding sites and SMFS can fully characterize a molecular interaction by quantitatively determining its energetic and kinetic parameters. The advantage of TREC-driven force spectroscopy is that a single functional molecule can be selected and targeted for force spectroscopy. We describe here a detailed protocol of performing AFM-based force spectroscopy guided by recognition imaging in order to map and characterize binding sites of membrane proteins reconstituted into lipid bilayers.

2. Experimental Design Based on AFM tapping mode in liquid [8,9], a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are then selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control [7]. The combined mode is an optimal tool for dynamic force probing on different pre-selected molecules, resulting in higher throughput when compared with force mapping. Moreover, the dynamics of force loading can be varied to elucidate the binding dynamics and map the interaction energy landscape [10]. A general functional principle of this technique is illustrated in Figure1. Combining the techniques of TREC imaging [11] and force spectroscopy [12] faces three major challenges: (i) A common technical issue during AFM measurements is thermal drift, which can lead to the loss of the protein position between two imaging scans or during force spectroscopy experiments. This drawback can be significantly improved by using a closed loop scanner (N9524A (Keysight, Santa Rosa, CA, USA): a multipurpose scanner with different nose cone cantilever modules, for acoustic or magnetic excitation), in which inductive sensors detect the deviations from the ideal movement and apply appropriate corrections to the piezo drive signal. (ii) It is mandatory to use the same cantilever for the entire combined TREC and force spectroscopy experiment. For an optimal force sensitivity a rather soft spring is required (spring constant 0.01–0.03 N/m) in conventional force spectroscopy [13], whereas for high-resolution dynamic imaging a stiffer spring (0.14–0.30 N/m) is preferred [11]. By identifying an acceptable compromise to satisfy both needs, a silicon nitride tip with a nominal spring constant of 0.03 N/m (MSNL levers (Bruker AFM Probes, Camarillo, CA, USA)) was used. Since such tips are not available with a magnetic coating (the standard for the magnetic excitation commonly used in TREC [11]), an acoustic excitation of the cantilever was applied instead. We thus optimized an alternative TREC mode for cantilevers with a small spring constant. (iii) In order to optimize the remaining drift with the usage of the closed loop scanner, constant environmental conditions were ensured. We strictly maintained a constant lab temperature (room temperature) Methods and Protoc. 2019, 2, 6 3 of 18 and started the measurements only when the system (sample, measurement buffer, and cantilever) appeared equilibrated. Methods Protoc. 2019, 2, x FOR PEER REVIEW 3 of 17

FigureFigure 1. 1.Experimental Experimental setup of of atomic atomic force force microscopy microscopy (AFM)-based (AFM)-based force force spectroscopy spectroscopy guided guided by byrecognition recognition imaging. imaging. (a) Receptors (a) Receptors reconstituted reconstituted into a intoplanar a planarlipid bilayer lipid spread bilayer on spread a on surface. a mica surface.The cantilever The cantilever is oscillated is oscillated by acoustical by acoustical excitation excitation over over the thesample sample surface. surface. (b ()b )Simultaneous Simultaneous topographicaltopographical and and recognitionrecognition (TREC) (TREC) imaging. imaging. The The oscillation oscillation amplitude amplitude is split is splitinto two into parts. two parts.The Theupper upper part part is taken is taken to toconstruct construct the the recognition recognition image, image, whereas whereas the the lower lower part part is isused used for for the the topographytopography image. image. Specific Specific interaction interaction ofof thethe tip-boundtip-bound ligand with with the the recept receptoror on on the the surface surface leads leads toto a a recognition recognition event event that that is is visible visible asas aa blackblack dotdot inin the recognition image image (red (red circles). circles). The The overlaid overlaid imageimage reveals reveals a a superposition superposition of of the the recognition recognition map map on on the the topographical topographical image. image. ( c(c)) The The cantilever cantilever is placedis placed over over the functionalthe functional receptor receptor (i.e., (i.e., molecules molecules that that generated generated recognition recognition signal signal (red (red circles)) circles)) and theand mode the mode is switched is switched to force to force spectroscopy. spectroscopy. In this In mode,this mode, the cantileverthe cantilever is repeatedly is repeatedly approached approached and and retracted from the sample surface, resulting in continuous recordings of force–distance cycles. A retracted from the sample surface, resulting in continuous recordings of force–distance cycles. A typical typical ligand-receptor unbinding event arising from force-induced ligand/receptor dissociation ligand-receptor unbinding event arising from force-induced ligand/receptor dissociation appears as a appears as a parabolic-shaped downward signal. Specific unbinding lengths above 10 nm can be parabolic-shaped downward signal. Specific unbinding lengths above 10 nm can be attributed to the attributed to the elasticity of the receptors and the lipid membrane. PEG: Polyethylene glycol. elasticity of the receptors and the lipid membrane. PEG: Polyethylene glycol.

InIn the the rare rare event event of of loss loss ofof unbindingunbinding signalssignals during force spectroscopy, spectroscopy, the the microscope microscope was was switchedswitched back back to to the the recognition recognition imagingimaging modemode and the cantilever was was readjusted readjusted to to the the protein protein moleculemolecule under under investigation. investigation. However, However, by by using using a positional a positional feedback feedback control control together together with awith careful a careful thermal stabilization protocol, the measurements were reasonably stable for at least 2 h thermal stabilization protocol, the measurements were reasonably stable for at least 2 h without loss of without loss of the binding site. the binding site. The procedures described in this protocol detail how this combined mode can be successfully The procedures described in this protocol detail how this combined mode can be successfully conducted. In the first part (Section 3.1), important references are provided on how proteoliposomes conducted. In the first part (Section 3.1), important references are provided on how proteoliposomes with containing membrane proteins can be prepared. The timing procedure depends on the chosen with containing membrane proteins can be prepared. The timing procedure depends on the chosen lipid composition and reconstituted protein, and can take up several days. The second part (Section lipid composition and reconstituted protein, and can take up several days. The second part (Section 3.2) 3.2) describes how the lipid bilayer with the incorporated receptors is formed on a surface for describes how the lipid bilayer with the incorporated receptors is formed on a surface for conducting conducting the AFM experiments (time: 15 min up to ~2 h). In the third part (Section 3.3), the AFM thecantilever AFM experiments is functionalized (time: with 15 min a cognitive up to ~2 ligand h). In theby utilizing third part a (Sectionheterobifunctional 3.3), the AFM crosslinker cantilever that is binds to both the amino-derivatized tip and the free amino-groups of the ligand. This procedure takes 2–3 days. The fourth part (Section 3.4) describes detailed procedures for application of this combined mode to characterize interaction sites at low lateral density. An additional chapter (4) reports the expected results and further applications. The proof-of-concept was shown for the quantitative

Methods Protoc. 2019Methods, 2, x FOR and PEER Protoc. REVIEW2019, 2, 6 4 of 17 4 of 18 characterization of the binding mechanism between mitochondrial membrane uncoupling protein 1 (UCP1) and itsfunctionalized inhibitor adenosine with triphosphate a cognitive (ATP). ligand Here by utilizing the binding a heterobifunctional dynamics as well as crosslinker the that binds to both interaction energythe amino-derivatizedlandscape was elucidated tip and [7,14] the free (Section amino-groups 4.1). In the of second the ligand. example, This the procedure takes 2–3 days. interaction between the bacterial translocation channel SecYEG and the cytoplasmic motor-protein The fourth part (Section 3.4) describes detailed procedures for application of this combined mode to SecA was characterized. The AFM-based force spectroscopy guided by recognition imaging was employed in ordercharacterize to localize interactionthe SecYEG comple sites atxes low and lateral map SecA density. binding An sites additional on suspended chapter lipid (4) reports the expected bilayers. Subsequently,results and the further interaction applications. forces Thebetween proof-of-concept SecYEG and wasSecA shown interaction for the quantitativewere characterization characterized. Finally,of the bindingSection 4.3 mechanism provides detailed between information mitochondrial on how membranethe data were uncoupling analyzed and protein 1 (UCP1) and its interpreted. inhibitor adenosine triphosphate (ATP). Here the binding dynamics as well as the interaction energy landscape was elucidated [7,14] (Section 4.1). In the second example, the interaction between the 2.1. Materials bacterial translocation channel SecYEG and the cytoplasmic motor-protein SecA was characterized.

• Acetal–PEGThe27–NHS AFM-based (available force by purchase spectroscopy from guidedH. Gruber, by recognitionJohannes Kepler imaging University was employed Linz in order to localize [15], Linz, theAustria). SecYEG Alternatively, complexes this and linker map can be SecA synthesized binding according sites on to suspended Reference [16]. lipid bilayers. Subsequently, CRITICALthe STEP interaction We recommend forces betweenpurchasing SecYEG monodisperse and SecA polyethylene interaction glycol were (PEG) characterized. linkers Finally, Section 4.3 from this providessource for detailedtwo reasons. information First, the onbenzaldehyde how the data function were analyzedis protected and by interpreted. an acetal- group for the binding to amino-functionalized tips via the activated carboxyl functionality of the N-hydroxysuccinimide2.1. Materials (NHS)-group. This elegantly precludes the possibility of bivalent interaction of the benzaldehyde function with adjacent amino-groups on the tip surface. Second, the linker is• suppliedAcetal–PEG in high-purity27–NHS 1-mg (available aliquots by ideally purchase suited from for use H. Gruber,in this protocol. Johannes Kepler University Linz [15], • DimethylsulfoxideLinz, anhydrous Austria). (>99.9%, Alternatively, (CH3)2SO (DMSO); this linker Sigma–Aldrich, can be synthesized St. Louis, MO, according USA, to Reference [16]. cat. no. 41640) CAUTIONCRITICAL DMSO STEP isWe harmful recommend upon inhalation purchasing and skin monodisperse absorption. Wear polyethylene glycol (PEG) appropriate gloves.linkers from this source for two reasons. First, the benzaldehyde function is protected by an • Sodium cyanoborohydrideacetal-group (NaCNBH for the binding3; Sigma–Aldrich, to amino-functionalized cat. no. 156159) tips viathe CAUTION activated carboxyl functionality NaCNBH3 is highlyof the toxic. N-hydroxysuccinimide Wear gloves and handle (NHS)-group. carefully. This elegantly precludes the possibility of bivalent • 3 Chloroform (>99.9%,interaction CHCl of; Sigma–Aldrich, the benzaldehyde cat. functionno. 288306) with adjacent CAUTION amino-groups Chloroform onis the tip surface. Second, highly volatile and harmful upon inhalation and skin absorption. Wear appropriate gloves and the linker is supplied in high-purity 1-mg aliquots ideally suited for use in this protocol. work under a fume hood. Handle only in glass vessels and glass dishes covered with a lid. • Dimethylsulfoxide anhydrous (>99.9%, (CH ) SO (DMSO); Sigma–Aldrich, St. Louis, MO, • Ethanol, absolute (>99.9%, C2H5OH; Sigma–Aldrich, cat. no. 24102).3 2 • (3-Aminopropyl)triethoxysilaneUSA, cat. no. 41640)(APTES, 99%,CAUTION Sigma–Aldrich,DMSO cat. is no. harmful 440140). upon Distill inhalation at low and skin absorption. temperature andWear store appropriate under argon gloves. in sealed crimp vials over silica gel at −20 °C to avoid polymerization. • Sodium cyanoborohydride (NaCNBH3; Sigma–Aldrich, cat. no. 156159) CAUTION NaCNBH3 • Ethanolamine hydrochloride (H2NC2H4OH; Sigma–Aldrich, cat. no. E6133). • 0.1 mM NaOH solutionis highly (in toxic. water, Wear Sigma–Aldrich, gloves and cat. handle no. 43617). carefully. • Molecular •sieves,Chloroform 3 Å (beads, (>99.9%,8–12 mesh; CHCl Sigma–Aldrich,3; Sigma–Aldrich, cat. no. cat.208574). no. 288306) CAUTION Chloroform is highly • Triethylamine >99.5%,volatile (C and2H5)3 harmfulN; Sigma–Aldrich, upon inhalation cat. no. 471283) and skin absorption.CAUTION Triethylamine Wear appropriate gloves and work is harmful uponunder inhalation a fume and hood. skin absorption, Handle only and in is glass volatile. vessels Wear and appropriate glass dishes gloves covered and with a lid. work under a fume hood. Store under argon and in the dark to avoid amine oxidation. • Ethanol, absolute (>99.9%, C2H5OH; Sigma–Aldrich, cat. no. 24102). • Milli-Q water (Merck Millipore, Burlington, MA, USA). • • Citric acid (Sigma–Aldrich,(3-Aminopropyl)triethoxysilane cat. no. 251275). (APTES, 99%, Sigma–Aldrich, cat. no. 440140). Distill at ◦ • Stock of membranelow temperatureproteins, reconstituted and store into under proteoliposomes. argon in sealed UCP1 and crimp SecYEG vials for over the silica gel at −20 C to examples shownavoid here. polymerization. • Ethylenediamine• Ethanolamine (EDA) derivates hydrochloride of the purine (H nucleotides2NC2H4OH; (EDA-ATP, Sigma–Aldrich, EDA-ADP cat. and no. EDA- E6133). AMP) (BioLog,• 0.1Bremen, mM Germany, NaOH solution cat. no. (inA072, water, A118) Sigma–Aldrich, or SecA protein cat.for tip no. coupling. 43617). • Na2SO4 (Sigma-Aldrich, cat. no. 238597). • Molecular sieves, 3 Å (beads, 8–12 mesh; Sigma–Aldrich, cat. no. 208574). • (NH4)2HPO4 (Sigma–Aldrich, cat. no. 215996). • 2-(N-morpholino)ethanesulfonic• Triethylamine >99.5%, acid (MES) (C2H (Sigma–Aldrich,5)3N; Sigma–Aldrich, cat. no. M3671). cat. no. 471283) CAUTION Triethylamine is • Tris (Sigma–Aldrich,harmful cat. uponno. 252859). inhalation and skin absorption, and is volatile. Wear appropriate gloves and work • Ethylene glycol-bis(underβ-aminoethyl a fume hood. ether)-N,N,N Store under′,N′-tetraacetic argon and acid in the(EGTA) dark (Sigma–Aldrich, to avoid amine cat. oxidation. no. E3889).• Milli-Q water (Merck Millipore, Burlington, MA, USA). • HEPES (Sigma–Aldrich, cat. no. H3375). • Citric acid (Sigma–Aldrich, cat. no. 251275). • HEPES potassium salt (K-HEPES) (Sigma–Aldrich, cat. no. H0527). • Sodium Chloride• Stock (NaCl) of (Sigma–Aldrich, membrane proteins, cat. no. reconstitutedS7653). into proteoliposomes. UCP1 and SecYEG for the examples shown here. • Ethylenediamine (EDA) derivates of the purine nucleotides (EDA-ATP, EDA-ADP and EDA-AMP) (BioLog, Bremen, Germany, cat. no. A072, A118) or SecA protein for tip coupling.

• Na2SO4 (Sigma-Aldrich, cat. no. 238597). Methods and Protoc. 2019, 2, 6 5 of 18

• (NH4)2HPO4 (Sigma–Aldrich, cat. no. 215996). • 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma–Aldrich, cat. no. M3671). • Tris (Sigma–Aldrich, cat. no. 252859). • Ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid (EGTA) (Sigma–Aldrich, cat. no. E3889). • HEPES (Sigma–Aldrich, cat. no. H3375). • HEPES potassium salt (K-HEPES) (Sigma–Aldrich, cat. no. H0527). • Sodium Chloride (NaCl) (Sigma–Aldrich, cat. no. S7653).

• Calcium Chloride (CaCl2) (Sigma–Aldrich, cat. no. C1016). • Magnesium Chloride (MgCl2) (Sigma–Aldrich, cat. no. M8266) • Potassium Chloride (KCl) (Sigma–Aldrich, cat. no. P9333) • Muscovite mica sheets (V-1 quality, Electron Microscopy Science, Hatfield, PA, USA, cat. no. 71855)

2.2. Equipment • Keysight 5500 AFM setup (or higher) equipped with PicoPlus box. • Closed-loop scanner (N9524A, Keysight). • AFM tapping nose cone for acoustic excitation (Keysight). • MSNL-lever (Bruker AFM Probes). • Inverted optical microscope. • Upright benchtop optical microscope for examining cantilevers and chips. • Active vibration isolation table. • Acoustic enclosure for the AFM. • AFM flow-through liquid .

• N2 gas bottle for drying cantilever chips. • Argon gas bottle for storing cantilever chips and chemicals properly without contamination. • pH meter. • Glass Pasteur pipettes and rubber bulbs for pH adjustment. • Analytical balance. • Fume hood. • Vacuum pump. • Vacuum desiccator. • Oven. • Plastic and glass Petri dishes (15 mm) with lid for tip functionalization and surface chemistry. • Tweezer. • Mechanical pipettors. • Pipette tips. • Hot plate stirrer. • Magnetic stir bars. • Glass beakers and Erlenmeyer flasks. • 4-cm glass crystallization dishes (Sigma–Aldrich, cat. no. BR455701). • Teflon block (3 × 1 × 0.5 cm). • Teflon reaction chamber (2 × 2 × 1.5 cm block indented with a cylindrical chamber 1-cm deep and 1.5 cm in diameter). • 12-well plates (Sigma–Aldrich, cat. no. CLS3513). • Vortex. • 50 µL and 500 µL Hamilton glass syringe. Methods and Protoc. 2019, 2, 6 6 of 18

Methods Protoc. 2019, 2, x FOR PEER REVIEW 4 of 17 • Gwyddion Analysis software (v. 2.49) or any other analysis software for scanning probe characterization of the binding mechanism between mitochondrial membrane uncoupling protein 1 microscopy (SPM) images, such as Pico Image (Keysight) or ImageJ. (UCP1) and its inhibitor adenosine triphosphate (ATP). Here the binding dynamics as well as the • interaction energy Originlandscape (Origin was 2016,elucidated Origin) [7,14] or any(Section other 4.1). scientific In the graphing second example, and data the analysis software. interaction between• Matlab the bacterial (2014 translocation or higher) + ch kspec19annel SecYEG analysis and routine the cytoplasmic (provided motor-protein by Keysight). SecA was characterized. The AFM-based force spectroscopy guided by recognition imaging was employed in order3. Procedure to localize the SecYEG complexes and map SecA binding sites on suspended lipid bilayers. Subsequently, the interaction forces between SecYEG and SecA interaction were characterized. Finally,3.1. Proteoliposome Section 4.3 provides Preparation detailed and Proteininformation Reconstitution. on how the Timedata were to Completion: analyzed and Several Days, Depending on interpreted. the Studied Biomolecule and Applied Protocol for Preparation Depending on the desired protein, which were to be reconstituted into proteoliposomes, different 2.1. Materials protocols were available [17,18]. For the examples shown here (see Section4 for expected results), • Acetal–PEGthe27–NHS following (available protocols by purchase can be consulted:from H. Gruber, Johannes Kepler University Linz [15], Linz, Austria). Alternatively, this linker can be synthesized according to Reference [16]. CRITICAL• STEPUCP1 We recommend in Escherichia purchasing coli polar monodisperse lipid extract polyethylene (PLE): Hilse glycol et al. [(PEG)19] and linkers Rupprecht et al. [20]. from this •sourceSecYEG for two in reasons. PLE: Knyazev First, the et be al.nzaldehyde [21,22]. function is protected by an acetal- group for the binding to amino-functionalized tips via the activated carboxyl functionality of the N-hydroxysuccinimide3.2. Surface Chemistry. (NHS)-group. Time This to Completion: elegantly 15precludes min up tothe ~2 possibility h, Depending of onbivalent the Studied Biomolecule interaction of the benzaldehyde function with adjacent amino-groups on the tip surface. Second, the linker 1.is suppliedPrepare in high-purity a freshly cleaved 1-mg aliquots mica byideally removing suited for the use upper in this layer protocol. of the quartz-like material with a • Dimethylsulfoxidecommercial anhydrous tape. (>99.9%, (CH3)2SO (DMSO); Sigma–Aldrich, St. Louis, MO, USA, cat. no. 41640) CRITICALCAUTION DMSO STEP isMake harmful sure upon to remove inhalation a closedand skin layer, absorption. to generate Wear an ultra-flat and clean appropriate gloves.surface for incubation of the protein-containing proteoliposomes. • Sodium cyanoborohydride2. Place the freshly (NaCNBH cleaved3; Sigma–Aldrich, mica on the AFM cat. sample no. 156159) plate and mountCAUTION a (flow-through) fluid cell. NaCNBH3 is highly toxic. Wear gloves and handle carefully. 3. Dilute the proteoliposome solution to a final concentration of 1 mg/mL lipid concentration with • Chloroform (>99.9%, CHCl3; Sigma–Aldrich, cat. no. 288306) CAUTION Chloroform is assay buffer. highly volatile and harmful upon inhalation and skin absorption. Wear appropriate gloves and work under4. a fumeAfter hood. short Handle vortexing, only in pipet glass the vessels proteoliposome and glass dishes solution covered into with the a fluid lid. cell and onto the clean and • Ethanol, absoluteflat (>99.9%, mica sheet. C2H5OH; Sigma–Aldrich, cat. no. 24102). • (3-Aminopropyl)triethoxysilane5. Depending onthe (APTES, lipid composition99%, Sigma–Aldrich, and the cat. protein, no. 440140). let the sampleDistill at incubate low for 10 min up to 2 h temperature andat store room under temperature argon in (RT). sealed After crimp that, vials lipid over bilayer silica batches gel at containing−20 °C to avoid the protein of choice should polymerization.be formed. OPTIONAL STEP Depending on the lipid composition (and their phase transition • 2 2 4 Ethanolamine hydrochloridetemperatures), (H youNC H mayOH; raise Sigma–Aldrich, the temperature cat. no. up E6133). to 60 ◦C during the incubation. • 0.1 mM NaOH solution (in water, Sigma–Aldrich, cat. no. 43617). • Molecular sieves, 3CRITICAL Å (beads, 8–12 STEP mesh;Make Sigma–Aldrich, sure to always cat. no. keep 208574). the sample moist to prevent evaporation of the

• Triethylamine >99.5%,liquid during(C2H5)3N; the Sigma–Aldrich, incubation. cat. no. 471283) CAUTION Triethylamine is harmful6. uponWash inhalation the sample and skin thoroughly absorption, with and assayis volatile. buffer Wear and appropriate add 500–600 glovesµL assay and buffer to the fluid cell work under a fumefor AFMhood. measurements.Store under argon and in the dark to avoid amine oxidation. • Milli-Q water (Merck Millipore, Burlington, MA, USA). • Citric acid3.3. (Sigma–Aldrich, AFM Tip Functionalization. cat. no. 251275). Time to Completion: 2–3 Days, Depending on the Chosen • Stock of membraneAminofunctionalization proteins, reconstituted Procedure into proteoliposomes. UCP1 and SecYEG for the examples shown here. The most common functionalization method is tethering the ligand on a silicon/silicon nitride • Ethylenediamine (EDA) derivates of the purine nucleotides (EDA-ATP, EDA-ADP and EDA- AMP) (BioLog,AFM Bremen, tip in Germany, a multiple-step cat. no. A0 crosslinking72, A118) or SecA procedure, protein for including tip coupling. the creation of reactive amino • Na2SO4 (Sigma-Aldrich,groups on the cat. chemically no. 238597). inert tip surface (Section 3.3.1), followed by the covalent binding of the • (NH4)2HPOcrosslinker4 (Sigma–Aldrich, (Section cat. 3.3.2 no. 215996).), and finally, coupling of the sensor molecule (Section 3.3.3). The inert • 2-(N-morpholino)ethanesulfonicwater soluble PEG-crosslinker acid (MES) (Sigma–Aldrich, was designed cat. as no. hetero-bifunctional M3671). [23], with one end being an • Tris (Sigma–Aldrich,N-hydroxysuccinimide cat. no. 252859). (NHS) group to bind free amino groups on the AFM tip and the other end • Ethylene glycol-bis(specificallyβ-aminoethyl designed to ether)-N,N,N react with′ the,N′-tetraacetic desired ligand acid (EGTA) for coupling. (Sigma–Aldrich, The latter cat. is available with different no. E3889). reactive molecules, such as an acetal-group to couple lysine residues (NH2), a malemide-group to • HEPES (Sigma–Aldrich, cat. no. H3375). couple thiol (SH)-containing molecules or a tris-nitrilotriacetic acid (NTA) group to bind His6-tagged • HEPES potassium salt (K-HEPES) (Sigma–Aldrich, cat. no. H0527). proteins. A general overview can be found in Reference [15]. • Sodium Chloride (NaCl) (Sigma–Aldrich, cat. no. S7653). For all tip functionalization procedures, the ligand density on the tip is adjusted such that on

average only one ligand has access to the receptors on the surfaces. Therefore, our tip functionalization design is also apt for single molecule studies at high receptor densities, see H. Gruber et al. [15]. Methods Protoc. 2019, 2, x FOR PEER REVIEW 7 of 17 Methods Protoc. 2019, 2, x FOR PEER REVIEW 4 of 17 characterization of theMethod binding A, with mechanism APTES in between the gas phase. mitochondrial membrane uncoupling protein 1 (UCP1) and its inhibitor1. CRITICAL adenosine STEP triphosphate Perform the (ATP). whole procedureHere the inbinding a well-ventilated dynamics hood. as well as the interaction energy2. landscapeWash cantilevers was inelucidated chloroform [7,14](3 × 5 min),(Section dry with 4.1). nitrogen In the gas, second continue example, with the nextthe step. interaction between3. theFlush bacterial desiccator translocation chamber (5 L) ch withannel argon SecYEG gas (through and the the cytoplasmic narrow opening motor-protein in the lid). Methods and Protoc. 2019, 2, 6 7 of 18 SecA was characterized.4. Place Thea tray AFM-based (caps from 1.5 force mL plastic spectroscopy Eppendorf guided tubes) with by 30recognition µL APTES andimaging another was tray with employed in order to10 localize µL triethylamine the SecYEG in the comple desiccator.xes and map SecA binding sites on suspended lipid bilayers. Subsequently,5. Place the cantileversinteraction in theforces desiccator between close toSecYEG the two trays. and Close SecA the interactionlid, incubate forwere 2 h. 3.3.1.6. Remove Aminofunctionalization the trays with APTES ofand the triethylamine, AFM Tip flush desiccator with argon. Incubate the tips characterized. Finally, Section 4.3 provides detailed information on how the data were analyzed and in argon for at least 2 days (“curing”). interpreted. In the following, two commonly used methods for aminofunctionalization of an AFM tip 7. PAUSE STEP Store under argon in a dust box for up to 3 weeks (preferably <1 week), if they (or sampleare not used substrates) immediately. are presented according to Reference [15,24,25]. While method A is more 2.1. Materials solid and leads to a very homogenous meshwork of amino groups, it requires some chemical expertise Method B, with Ethanolamine Hydrochloride in DMSO. • Acetal–PEGand27–NHS experience (available of the by operatorpurchase duefrom to H. a reactionGruber, Johannes step in the Kepler gas phaseUniversity and theLinz volatility of both APTES 1. Wash cantilevers in chloroform (3 × 5 min), dry with nitrogen gas, continue with the next step. [15], Linz,and Austria).2. triethylamine.Dissolve Alternatively, 3.3 g ethanolamine Moreover, this linker hydrochloride APTES can be silanization synthesized in 6.6 mL isaccordingDMSO, very cover critical to withReference with lid, heat respect [16]. to ~70 to °C the for freshness of APTES CRITICAL(hydrolyzes STEPcomplete We recommend very dissolution, fast in purchasing let air) cool and to room exclusion monodisperse temperat ofure. air. polyethyleneIn For addition, unexperienced a magnetic glycol (PEG) stirrer users, linkerscan method be added B in liquid phase is from this highlysourceto for recommended.the twosolution reasons. to speed First, Both up the the methods dissolving benzaldehyde are process. fully function satisfactory is protected with respectby an acetal- to the attachment of ligands group for singlethe3. bindingImmerse molecule to amino-functionalizeda Teflon force block spectroscopy for the cantilevers tips experiments. via the in thactivatede center Parallel ofcarboxyl the dish macroscopic functionality and add 3–4 assays Åof molecularthe on silicon nitride chips N-hydroxysuccinimidesieves beads (NHS)-group. to cover the surrounding This eleg antlyarea (~25% precludes of the total the volume possibility of the liquid).of bivalent showed that APTES appeared to yield a slightly lower surface density of amino groups [25]. interaction of4. thePut benzaldehyde the dish into afunction desiccator with (or adjavacuumcent cham amino-groupsber) and degas on thethe tipsolution surface. and Second, the molecular the linker is suppliedMethodsieves inby high-purity A,applying with an APTES aspirator 1-mg inaliquots vacuum the gas ideally (or phase. similar) suited for for ~30 use min. in this protocol. 5. Place cantilevers on the Teflon block, cover with lid, incubate at room temperature overnight. • Dimethylsulfoxide anhydrous (>99.9%, (CH3)2SO (DMSO); Sigma–Aldrich, St. Louis, MO, USA, 6. Wash cantilevers in DMSO (3 × 1 min) and ethanol (3 × 1 min). cat. no. 41640)1.7. Dry CRITICALCAUTION with a gentle DMSOstream STEP ofPerformis nitrogen harmful theor upon argon whole inhalationgas. procedure and inskin a well-ventilatedabsorption. Wear hood. appropriate2. 8.gloves. Wash PAUSE cantilevers STEP Store in under chloroform argon in a (3 dust× 5box min), for up dry to 3 with weeks nitrogen (preferably gas, <1 week), continue if they with the next step. • 3 Sodium cyanoborohydride3. Flushare not desiccatorused immediately. (NaCNBH chamber ; Sigma–Aldrich, (5 L) with argon cat. gas no. (through 156159) the narrow CAUTION opening in the lid). NaCNBH3 is highly toxic. Wear gloves and handle carefully. 4.3.3.2.Place NHS–PEG a tray27–Acetal (caps Coupling from 1.5 mL plastic Eppendorf tubes) with 30 µL APTES and another tray with • Chloroform (>99.9%, CHCl3; Sigma–Aldrich, cat. no. 288306) CAUTION Chloroform is 10 µL triethylamine in the desiccator. highly volatile1. andDissolve harmful 1 portion upon of inhalation Acetal–PEG an27d–NHS skin (1absorption. mg) in chloroform Wear appropriate (0.5 mL), transfer gloves the and solution work under5. a fumePlaceinto hood.the the reaction cantileversHandle chamber, only in inadd theglass trieth desiccator vesselsylamine and (30 close glassµL) and to dishes themix twoby covered pipetting. trays. with Close a lid. the lid, incubate for 2 h. 2. Immediately place cantilever(s) in the reaction chamber, cover the chamber and incubate for 2 • Ethanol, absolute6. Remove (>99.9%, the C2H trays5OH; with Sigma–Aldrich, APTES and cat. triethylamine, no. 24102). flush desiccator with argon. Incubate the tips h. • (3-Aminopropyl)triethoxysilane (APTES, 99%, Sigma–Aldrich, cat. no. 440140). Distill at low 3. inWash argon with forchloroform at least (3 2 × days10 min) (“curing”). and dry with nitrogen or argon gas. temperature and store under argon in sealed crimp vials over silica gel at −20 °C to avoid 7.4. PAUSE STEP STEP StoreStore cantilever(s) under for argon up to inseveral a dust months box under for up argon, to 3 or weeks continue (preferably with next <1 week), if they polymerization. arestep not(Section used 3.3.3). immediately. • Ethanolamine hydrochloride (H2NC2H4OH; Sigma–Aldrich, cat. no. E6133). • 0.1 mM NaOH3.3.3. solution Coupling (in of water, Interacting, Sigma–Aldrich, Corresponding cat. Biomolecule no. 43617). to the Free Acetal-End Method B, with Ethanolamine Hydrochloride in DMSO. • Molecular sieves, 3 Å (beads, 8–12 mesh; Sigma–Aldrich, cat. no. 208574). 1. Immerse the cantilevers functionalized with NHS–PEG27–Acetal cross linker for 10 min in 1% • Triethylamine >99.5%, (C2H5)3N; Sigma–Aldrich, cat. no. 471283) CAUTION Triethylamine 1. Wash(w/v) citric cantilevers acid (in water). in chloroform (3 × 5 min), dry with nitrogen gas, continue with the next step.

is harmful upon2. Wash inhalation the cantilevers and skin in Milli-Qabsorption, water (3and × 5 is min) volatile. and dry Wear with appropriatenitrogen or argon gloves gas. and ◦ work under2.3. a fumeDissolveFreshly hood. prepare 3.3Store g a ethanolamineunder 1 M solution argon anof d hydrochloridesodium in the cydarkanoborohydride to avoid in 6.6 amine mL (with DMSO, oxidation. 20 mMcover NaOH) with from lid,the heat to ~70 C for • Milli-Q water (Merckcompletefollowing Millipore, components: dissolution, Burlington, 13 mg let NaCNBH cool MA, to USA). room3, 20 µL temperature. 100 mM NaOH, Inand addition, 180 µL Milli-Q a magnetic water. stirrer can be added • Citric acid (Sigma–Aldrich,to theCRITICAL solution cat. STEP no. to speedNaCNBH251275). up 3 is the highly dissolving toxic. Wear process. gloves and handle with care.

• Stock of membrane3.4. ImmersePlace proteins,the AFM a Teflon chips reconstituted in block a radial for arrangement,into the proteoliposomes. cantilevers with the intips the UCP1in the center center and ofSecYEGand the facing dish for upwards, the and add on 3–4 Å molecular Parafilm in a polystyrene Petri dish. examples shown here. 5. sievesPipet 100 beads µL protein to cover solution the (1–2 surrounding µM) carefully area onto (~25% the tip of of each the cantilever total volume so that ofthey the are liquid). • Ethylenediamine4. Putextended (EDA) the dishintoderivates the into protein of a desiccatorthe drop. purine In the (ornucleotides first vacuum example (EDA-ATP, chamber)shown here, EDA-ADP andan EDA degas derivate and the EDA- of solution different and the molecular AMP) (BioLog, Bremen, Germany, cat. no. A072, A118) or SecA protein for tip coupling. sievespurine nucleotides by applying was an coupled aspirator to the vacuum AFM tip (orto study similar) their for interaction ~30 min. with uncoupling • Na2SO4 (Sigma-Aldrich,proteins. Incat. the no. second 238597). example, the ATP motor-protein SecA was attached onto the AFM tip in 5. Place cantilevers on the Teflon block, cover with lid, incubate at room temperature overnight. • (NH4)2HPO4 (Sigma–Aldrich,order to investigate cat. no.its interaction 215996). with the bacterial translocon SecYEG. • × × 2-(N-morpholino)ethanesulfonic6. Wash cantilevers acid in DMSO(MES) (Sigma–Aldrich, (3 1 min) and cat. ethanol no. M3671). (3 1 min). • Tris (Sigma–Aldrich,7. Dry with cat. no. a gentle 252859). stream of nitrogen or argon gas. • Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma–Aldrich, cat. 8. PAUSE STEP Store under argon in a dust box for up to 3 weeks (preferably <1 week), if they no. E3889). • HEPES (Sigma–Aldrich,are not usedcat. no. immediately. H3375). • HEPES potassium salt (K-HEPES) (Sigma–Aldrich, cat. no. H0527). • Sodium Chloride3.3.2. NHS–PEG (NaCl) (Sigma–Aldrich,27–Acetal Coupling cat. no. S7653).

1. Dissolve 1 portion of Acetal–PEG27–NHS (1 mg) in chloroform (0.5 mL), transfer the solution into the reaction chamber, add triethylamine (30 µL) and mix by pipetting. 2. Immediately place cantilever(s) in the reaction chamber, cover the chamber and incubate for 2 h. 3. Wash with chloroform (3 × 10 min) and dry with nitrogen or argon gas. 4. PAUSE STEP Store cantilever(s) for up to several months under argon, or continue with next step (Section 3.3.3). Methods Protoc. 2019, 2, x FOR PEER REVIEW 4 of 17 characterization of the binding mechanism between mitochondrial membrane uncoupling protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP). Here the binding dynamics as well as the interaction energy landscape was elucidated [7,14] (Section 4.1). In the second example, the interaction between the bacterial translocation channel SecYEG and the cytoplasmic motor-protein SecA was characterized. The AFM-based force spectroscopy guided by recognition imaging was employed in order to localize the SecYEG complexes and map SecA binding sites on suspended lipid bilayers. Subsequently, the interaction forces between SecYEG and SecA interaction were Methods Protoc. 2019, 2, x FOR PEER REVIEW 7 of 17 characterized. Finally, Section 4.3 provides detailed information on how the data were analyzed and interpreted. Method A, with APTES in the gas phase. 2.1. Materials Methods1. and CRITICAL Protoc. 2019 STEP, 2, 6 Perform the whole procedure in a well-ventilated hood. 8 of 18 • Acetal–PEG2.27 –NHSWash (available cantilevers by in chloroformpurchase from(3 × 5 min),H. Gruber, dry with Johannes nitrogen gas,Kepler continue University with the Linznext step. [15], Linz, Austria).3. Flush Alternatively, desiccator chamber this linker (5 L) with can argonbe synthesized gas (through according the narrow to opening Reference in the [16]. lid). 4. Place a tray (caps from 1.5 mL plastic Eppendorf tubes) with 30 µL APTES and another tray with CRITICAL3.3.3. STEP Coupling We recommend of Interacting, purchasing Corresponding monodisperse Biomoleculepolyethylene glycol to the (PEG) Free Acetal-End linkers 10 µL triethylamine in the desiccator. from this source for two reasons. First, the benzaldehyde function is protected by an acetal- 1.5. ImmersePlace the cantilevers the cantilevers in the desiccator functionalized close to the two with trays. NHS–PEG Close the 27lid,–Acetal incubate crossfor 2 h. linker for 10 min in 1% group for the6. binding(Removew/v) to citric the amino-functionalized trays acid with (in APTES water). and tips triethylamine, via the activated flush desiccator carboxyl with functionality argon. Incubate of the the tips N-hydroxysuccinimidein argon for(NHS)-group. at least 2 days (“curing”).This eleg antly precludes the possibility of bivalent 2. Wash the cantilevers in Milli-Q water (3 × 5 min) and dry with nitrogen or argon gas. interaction of7. the benzaldehyde PAUSE STEP Storefunction under with argon adja incent a dust amino-groups box for up to 3 onweeks the (preferablytip surface. <1 Second, week), if they the linker 3.is suppliedFreshlyare not in used high-purity prepare immediately. a 1-mg 1 M solutionaliquots ideally of sodium suited cyanoborohydridefor use in this protocol. (with 20 mM NaOH) from the • µ µ DimethylsulfoxidefollowingMethod anhydrous B, with components: Ethanolamine(>99.9%, (CH 13 Hydrochloride3) mg2SO NaCNBH(DMSO); in Sigma–Aldrich,DMSO.3, 20 L 100 mM St. Louis, NaOH, MO, and USA, 180 L Milli-Q water. cat. no. 41640) CAUTION DMSO is harmful upon inhalation and skin absorption. Wear 1. WashCRITICAL cantilevers in STEP chloroformNaCNBH (3 × 5 min),3 is highly dry with toxic. nitrogen Wear gas, glovescontinue andwith handlethe next step. with care. appropriate4.2. gloves. PlaceDissolve the 3.3 AFMg ethanolamine chips in hydrochloride a radial arrangement, in 6.6 mL DMSO, with cover the with tips lid, in heat the to center ~70 °C andfor facing upwards, • Sodium cyanoborohydrideoncomplete Parafilm dissolution, in(NaCNBH a polystyrene let cool3; toSigma–Aldrich, room Petri temperat dish.ure. cat. In addition,no. 156159) a magnetic stirrer CAUTION can be added NaCNBH3 is highlyto the toxic. solution Wear to speed gloves up and the dissolvinghandle carefully. process. 5. Pipet 100 µL protein solution (1–2 µM) carefully onto the tip of each cantilever so that they are • Chloroform3. (>99.9%, Immerse CHCl a Teflon3; Sigma–Aldrich, block for the cantilevers cat. no. in288306) the center of CAUTION the dish and Chloroform add 3–4 Å molecular is extended into the protein drop. In the first example shown here, an EDA derivate of different highly volatile andsieves harmful beads to upon cover inhalation the surrounding and skin area absorption.(~25% of the totalWear volume appropriate of the liquid). gloves and

work under4. a fumepurinePut thehood. dish nucleotides Handle into a desiccator only was in glass (or coupled vacuum vessels tocham and the ber)glass AFM and dishes degas tip covered to the study solution with their and a lid. the interaction molecular with uncoupling sieves by applying an aspirator vacuum (or similar) for ~30 min. • Ethanol, absoluteproteins. (>99.9%, InC2 theH5OH; second Sigma–Aldrich, example, thecat. ATPno. 24102). motor-protein SecA was attached onto the AFM tip in 5. Place cantilevers on the Teflon block, cover with lid, incubate at room temperature overnight. • order to investigate its interaction with the bacterial translocon SecYEG. (3-Aminopropyl)triethoxysilane6. Wash cantilevers in (APTES, DMSO (3 99%,× 1 min) Sigma–Aldrich, and ethanol (3 ×cat. 1 min). no. 440140). Distill at low temperature6.7. andAddDry store with 2 µ underaL gentle of the argon stream freshly in of senitrogen preparedaled crimp or argon 1 vials M gas. NaCNBH over silica3 stock gel at solution −20 °C to to avoid the protein solution droplet, polymerization.8. mix PAUSE carefully STEP by Store pipetting, under argon cover in a dust with box a lid,for up and to 3 incubate weeks (preferably for 1 h. <1 week), if they • Ethanolamine hydrochlorideare not used immediately. (H2NC2H4 OH; Sigma–Aldrich, cat. no. E6133). 7. OPTIONAL STEP Dissolve 3 pellets NaOH in 500 mL water, add the unused NaCNBH3 solution, • 0.1 mM NaOH solution (in water, Sigma–Aldrich, cat. no. 43617). mix, wait overnight, pour into the drain, and flush with tap water. • Molecular sieves,3.3.2. NHS–PEG 3 Å (beads,27–Acetal 8–12 Couplingmesh; Sigma–Aldrich, cat. no. 208574). 8. To passivate unreached aldehyde groups, add 5 µL of ethanolamine solution (1 M, pH 8.0) to the • Triethylamine1. >99.5%,Dissolve (C 1 2Hportion5)3N; Sigma–Aldrich,of Acetal–PEG27–NHS cat. no.(1 mg) 471283) in chloroform CAUTION (0.5 mL), Triethylamine transfer the solution is harmful upondropinto inhalation the on reaction the and cantilever(s), chamber, skin absorption, add trieth mixylaminecautiously and is (30volatile. µL) by and pipetting, Wear mix by appropriate pipetting. cover with gloves a lid, and and incubate for 10 min. work under9. 2.a fumeWashImmediately hood. in PBS Store place or under anycantilever(s) otherargon bufferinan thed in reaction ofthe choice dark chamber, to (3 avoid× cover5 min).amine the chamberoxidation. and incubate for 2 h. • Milli-Q water (Merck Millipore, Burlington, MA, USA). 10.3. MountWash with cantilever chloroform in (3 AFM× 10 min) setup and ifdry used with immediatelynitrogen or argon for gas. experiments. • Citric acid (Sigma–Aldrich, cat. no. 251275). 4. PAUSE STEP STEP Store cantilever(s) for up to several months under argon, or continue with next ◦ • If not used immediately, store cantilever in a 24-well plate under PBS at 4 C Stock of membranestep (Sectionproteins, 3.3.3). reconstituted into proteoliposomes. UCP1 and SecYEG for the examples shownfor here. 1–2 weeks (depending on the stability of the conjugated biomolecule). • Ethylenediamine3.3.3. Coupling (EDA)CRITICAL derivates of Interacting, STEP of the CorresponTransfer purineding thenucleotides Biomolecule functionalized (EDA-ATP, to the Free AFM EDA-ADPAcetal-End cantilevers and toEDA- PBS buffer and then to the AMP) (BioLog, Bremen, Germany, cat. no. A072, A118) or SecA protein for tip coupling. 1. AFMImmerse always the cantilevers very rapidly functionalized so that with they NHS–PEG do not27–Acetal dry (withincross linker 20 for s). 10 Otherwise,min in 1% the conjugated • Na2SO4 (Sigma-Aldrich,biomolecule(w/v) citric cat. acid no. could(in 238597). water). start to denaturate and lose its bio-functionality. • (NH4)2HPO42. (Sigma–Aldrich, Wash the cantilevers cat. no. in Milli-Q215996). water (3 × 5 min) and dry with nitrogen or argon gas. • 2-(N-morpholino)ethanesulfonic3.4.3. AFMFreshly Measurement. prepare a 1acid M Time solution (MES) to Completion: (Sigma–Aldrich,of sodium cy ~4anoborohydride h upcat. to no. 1 Day,M3671). (with Depending 20 mM NaOH) on the from Studied the Biomolecule and the • Tris (Sigma–Aldrich,Desiredfollowing Data cat. Throughput components:no. 252859). 13 mg NaCNBH3, 20 µL 100 mM NaOH, and 180 µL Milli-Q water. • Ethylene glycol-bis( CRITICALβ-aminoethyl STEP ether)-N,N,N NaCNBH3 is highly′,N′-tetraacetic toxic. Wear acid gloves (EGTA) and handle (Sigma–Aldrich, with care. cat. no. E3889).1. 4. SwitchPlace the onAFM the chips AFM in a equippedradial arrangement, with a with PicoPlus the tips 5500in the boxcenter (Keysight and facing upwards, Technologies) on and the AFM Parafilm in a polystyrene Petri dish. • HEPES (Sigma–Aldrich,controlling cat. softwareno. H3375). (PicoView, usable versions starting from 1.12 until 1.20). 5. Pipet 100 µL protein solution (1–2 µM) carefully onto the tip of each cantilever so that they are • HEPES potassium2. Switch salt (K-HEPES) on the inverted (Sigma–Aldrich, optical microscope cat. no. H0527). and the related controlling software. extended into the protein drop. In the first example shown here, an EDA derivate of different • Sodium Chloride3. Mountpurine (NaCl) nucleotides the (Sigma–Aldrich, AFM was liquid coupled cellcat. withno.to the S7653). theAFM sample tip to study on the their AFM interaction stage fromwith uncoupling the bottom. 4. Mountproteins. theIn the AFM second tip example, derivatized the ATP with motor- theprotein corresponding, SecA was attached interacting onto the molecule AFM tip in onto the cantilever holderorder to (tappinginvestigate noseits interaction cone), slotwith thethe bacterial holder translocon into the closed-loop SecYEG. scanner and assemble everything onto the AFM stage from the top. CRITICAL STEP Ensure that the Z stepper motors are sufficiently retracted to prevent crashing the cantilever into the bottom to the fluid cell and onto the sample. CRITICAL STEP Perform these steps rapidly. A drop of the assay buffer should always remain on the cantilever chip during transfer and should not be allowed to dry out. CRITICAL STEP This experiment works only with closed-loop scanners (e.g., N952A, Keysight Technologies) and the tip acoustically excited. Make sure to connect the cable for the closed-loop scanner on the AFM stage and switch on the closed-loop option in the software. Moreover, use the right nose cone (for acoustic excitation, “tapping nose”). 5. Align the spot at the free end of the cantilever and maximize the sum into the photodetector. 6. Bring the cantilever close to contact with the sample as follows: Using a wide-field imaging mode and the manual controls, locate the shadow of the cantilever by adjusting the AFM head Methods Protoc. 2019, 2, x FOR PEER REVIEW 4 of 17 characterization of the binding mechanism between mitochondrial membrane uncoupling protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP). Here the binding dynamics as well as the interaction energy landscape was elucidated [7,14] (Section 4.1). In the second example, the interaction between the bacterial translocation channel SecYEG and the cytoplasmic motor-protein SecA was characterized. The AFM-based force spectroscopy guided by recognition imaging was employed in order to localize the SecYEG complexes and map SecA binding sites on suspended lipid bilayers. Subsequently, the interaction forces between SecYEG and SecA interaction were characterized. Finally, Section 4.3 provides detailed information on how the data were analyzed and interpreted.

2.1. Materials Methods and Protoc. 2019, 2, 6 9 of 18

• Acetal–PEG27–NHS (available by purchase from H. Gruber, Johannes Kepler University Linz [15], Linz, Austria).position Alternatively, until the this cantilever linker can is be directly synthesized in the according light path. to Reference Using the [16]. AFM software, slowly drive CRITICAL STEPthe We Z stepperrecommend motors purchasing of the AFMmonodisperse head down polyethylene until the glycol cantilever (PEG) is linkers within the focal range of the from this sourcemicroscope for two reasons. objective First, and the bringbenzaldehyde it into focus. function It might is protected be necessary by an acetal- to make fine adjustments in group for the binding to amino-functionalized tips via the activated carboxyl functionality of the the position of the laser spot at this time. N-hydroxysuccinimide (NHS)-group. This elegantly precludes the possibility of bivalent interaction7. of theAllow benzaldehyde 15 min function up to1 with h for adja thecent system amino-groups to equilibrate, on the tip surface. until the Second, vertical offset of the laser the linker is suppliedspot onin high-purity the photodiode 1-mg aliquots no longer ideally drifts. suited Tryfor use to in keep this theprotocol. labtemperature constant during • Dimethylsulfoxidethe anhydrous whole experiment. (>99.9%, (CH3)2SO (DMSO); Sigma–Aldrich, St. Louis, MO, USA, cat. no. 41640) CRITICALCAUTION DMSO STEP isPerform harmful thisupon step inhalation verythoughtfully and skin absorption. and considerate. Wear A stable system appropriate gloves.with as less thermal drift (in x–y directions) as possible is very important for the upcoming • Sodium cyanoborohydrideexperimental (NaCNBH steps. 3; Sigma–Aldrich, cat. no. 156159) CAUTION NaCNBH3 is highly toxic. Wear gloves and handle carefully. • Chloroform3.4.1. (>99.9%, TREC CHCl Imaging:3; Sigma–Aldrich, Protein Distribution cat. no. 288306) and Functionality CAUTION Chloroform is highly volatile and harmful upon inhalation and skin absorption. Wear appropriate gloves and work under1. a fumeChoose hood. AFM Handle tapping only in mode glass invessels the software. and glass dishes covered with a lid. • Ethanol, absolute2. Select (>99.9%, the C following2H5OH; Sigma–Aldrich, channels for cat. recognition no. 24102). imaging: height, recognition image channel • (3-Aminopropyl)triethoxysilane(commonly referred (APTES, as AUX99%, channel),Sigma–Aldrich, amplitude, cat. no. and 440140). phase—both Distill at for low trace and retrace. temperature3. andRun store a tuningunder argon curve in (0–50 sealed kHz) crimp to vials set aover proper silica frequencygel at −20 °C for to the avoid acoustic excitation of the polymerization.cantilever [7]. To achieve an adequate driving amplitude, chose the second frequency level • 2 2 4 Ethanolamine hydrochloride(f ~ 21 kHz) for(H NC recognitionH OH; Sigma–Aldrich, imaging instead cat. no. of the E6133). base resonance frequency (f ~ 10 kHz) for the • 0.1 mM NaOH solution (in water, Sigma–Aldrich, cat. no. 43617). MSNL lever. • Molecular sieves, 3 Å (beads, 8–12 mesh; Sigma–Aldrich, cat. no. 208574). 4. To engage, choose the following settings: • Triethylamine >99.5%, (C2H5)3N; Sigma–Aldrich, cat. no. 471283) CAUTION Triethylamine is harmful upon inhalation and skin absorption, and is volatile. Wear appropriate gloves and a. Amplitude setpoint ~4 V, adjust with drive. work under a fume hood. Store under argon and in the dark to avoid amine oxidation. • Milli-Q water (Merckb. Millipore,Stop at amplitude Burlington, reduction MA, USA). of 80%. • Citric acid5. (Sigma–Aldrich,Start the imaging cat. no. 251275). and optimize the AFM recognition imaging parameters (amplitude setpoint, • Stock of membraneoscillation proteins, frequency, reconstituted feedback into proteoliposomes. gains). This is partiallyUCP1 and a trial-and-errorSecYEG for the process and will differ examples shown here. between the samples, as well as the coupled biomolecule to the tip. Topographs should appear • Ethylenediamine (EDA) derivates of the purine nucleotides (EDA-ATP, EDA-ADP and EDA- similar regardless of scanning direction (trace and retrace). The corresponding recognition events, AMP) (BioLog, Bremen, Germany, cat. no. A072, A118) or SecA protein for tip coupling. displayed in the AUX channel, should match with topographic features. A brief description of • Na2SO4 (Sigma-Aldrich, cat. no. 238597). • (NH4)2HPO4 (Sigma–Aldrich,some key parameters cat. no. 215996). follows, along with suggested value ranges and can be found in more detail • 2-(N-morpholino)ethanesulfonicin Preiner et al. [acid26]: (MES) (Sigma–Aldrich, cat. no. M3671). • Tris (Sigma–Aldrich, cat. no. 252859). • Ethylene glycol-bis(a. β-aminoethylThe proper ether)-N,N,N chosen oscillation′,N′-tetraacetic amplitude acid (EGTA) is the (Sigma–Aldrich, most prerequisite cat. to achieve reliable no. E3889). recognition events. If the linker is stretched too less at low oscillation amplitudes or, • HEPES (Sigma–Aldrich,alternatively, cat. no. H3375). if the receptor—ligand bond is broken at each oscillation cycle when using too • HEPES potassium salt large(K-HEPES) amplitudes, (Sigma–Aldrich, no pronounced cat. no. H0527). and stable recognition events are observed. The range of • Sodium Chloride (NaCl)appropriate (Sigma–Aldrich, amplitudes cat. no. for S7653). recognition imaging is determined by the effective linker length (including the position and the length of the molecule attached to the linker), and therefore sharply localized. Run an amplitude–distance curve and test different amplitude setpoints (while keeping the ratio of amplitude setpoint and free amplitude constant) to find the best working amplitude for achieving pronounced recognition signals. The value varies usually between 1.5 and 2 V. b. Additionally, the variation of the amplitude can be also used as a specificity proof, besides the widely used tip/surface block (Figure2b) without perturbation of the receptor–ligand system. This procedure is based on the physical property of the signal transduction of the recognition process. Vary the amplitude to very high (~3–4 V) and very low (<1 V) values. If the recognition spots disappear at this value and appear again at the proper set value (~2 V), it will give the user the proof for specific interaction events between the chosen receptor–ligand pair. c. The oscillation frequency has direct influence on the topographical crosstalk in the recognition signal. Adjust the value always lower than the resonance frequency (2nd order) Methods and Protoc. 2019, 2, 6 10 of 18

of the cantilever at tip/surface contact. For lower driving frequencies, the topographical perturbation has more time to decay until the recognition signal is detected. Methods Protoc. 2019, 2, x FORd. PEEROptimize REVIEW the feedback gains to allow the AFM tip to accurately 4 oftrack 17 the surface. Increase characterization of the bindingthe gainmechanism to the pointbetween at whichmitochondrial feedback membrane oscillation uncoupling noise is observed protein 1 in the AFM topography (UCP1) and its inhibitor adenosineimage. Slightly triphosphate reduce (ATP). the gainHere tothe remove binding this dynamics effect. as well as the interaction energy landscape was elucidated [7,14] (Section 4.1). In the second example, the 6. Scan the sample with above suggested parameters and find a good sample position, interaction between the bacterial translocation channel SecYEG and the cytoplasmic motor-protein more precisely an area with single proteins incorporated in the lipid membrane and with clearly SecA was characterized. The AFM-based force spectroscopy guided by recognition imaging was employed in order todefined localize recognitionthe SecYEG comple spots whichxes and match map SecA with binding the topography. sites on suspended For doing lipid so, first scan a bigger area bilayers. Subsequently,(~5–10 theµ m)interaction to find single,forces bigbetween bilayer SecYEG batches and graduallySecA interaction zoom-in were to visualize the proteins in characterized. Finally,the Section membrane 4.3 provides and to detailed study their information recognition on how with the the data derivatized were analyzed molecule and on the tip (500–700 nm interpreted. scan size). 7. OPTIONAL STEP Change the sample and/or adjust the incubation time, lipid concentration, 2.1. Materials temperature, etc., to optimize the formation of single, big lipid bilayer batches on mica with the • Acetal–PEG27–NHSincorporated (available moleculeby purchase homogenously from H. Gruber, distributed Johannes Kepler (Figure University2a). If no recognitionLinz signals appear [15], Linz, Austria).after Alternatively, adjusting all this parameters, linker can be change synthesized the tip. according to Reference [16]. CRITICAL8. STEPAfter We findingrecommend a suitable purchasing area monodisperse with a size of polyethylene 500–700 nm glycol2 that (PEG) covered linkers by a complete membrane from this sourcebatch for two check reasons. whether First, thethe membranebenzaldehyde proteins function are is protected mostly visualized by an acetal- as single, round-shaped group for the binding to amino-functionalized tips via the activated carboxyl functionality of the structures showing specific interaction (Figure2a). Then start scanning this area several times N-hydroxysuccinimide (NHS)-group. This elegantly precludes the possibility of bivalent interaction of theup benzaldehyde and down withoutfunction with changing adjacent any amino-groups parameters. on As the soon tip surface. as no Second, thermal drift occurs anymore, the linker is suppliedand the in high-purity subsequent 1-mg scans aliquots show ideally no change suited for in termsuse in this of positionprotocol. displacement/shift, stop the • Dimethylsulfoxidemeasurement anhydrous (>99.9%, and switch (CH3 from)2SO (DMSO); tapping Sigma–Aldrich, to contact mode. St. Louis, MO, USA, cat. no. 41640) CRITICALCAUTION DMSO STEP isBefore harmful switching upon inhalation to contact and mode, skin absorption. make sure Wear that the thermal drift is as appropriate gloves.minimal as possible, since the subsequent force spectroscopy experiment is carried out “blindly” • Sodium cyanoborohydridewithout a visual (NaCNBH control3; ofSigma–Aldrich, the correct cantilever cat. no. position156159) on the CAUTION depicted molecule. NaCNBH3 is highly toxic. Wear gloves and handle carefully. • Chloroform3.4.2. (>99.9%, Force CHCl Spectroscopy:3; Sigma–Aldrich, Dynamics cat. andno. Kinetics288306) of InteractingCAUTION PartnersChloroform is highly volatile and harmful upon inhalation and skin absorption. Wear appropriate gloves and work under1. a fumeAfter hood. changing Handle to only contact in glass mode, vessels position and glass the dishes cantilever covered ona with selected a lid. recognition spot (functional • Ethanol, absolutemolecule (>99.9%, showingC2H5OH; Sigma–Aldrich, strong binding cat. in TRECno. 24102). mode) using positional feedback control. • (3-Aminopropyl)triethoxysilane2. Start the force (APTES, spectroscopy 99%, Sigma–Aldrich, experiment by cat. recording no. 440140). force Distill distance at low curves directly on the temperature anddepicted store under spot argon using in well-adjusted sealed crimp vials parameters over silica (pulling gel at − speed,20 °C to hold avoid times, etc.) (Figure1c). polymerization.Since binding/unbinding is a record at least 500–1000 curves per pulling speed. • 2 2 4 Ethanolamine3. hydrochlorideOPTIONAL (H STEPNC HInOH; order Sigma–Aldrich, to probe the dynamicscat. no. E6133). and kinetics of the receptor-ligand interaction • 0.1 mM NaOH solution (in water, Sigma–Aldrich, cat. no. 43617). vary the pulling speed, and thus, loading rate after recording a data set of 500–1000 curves. • Molecular sieves, 3 Å (beads, 8–12 mesh; Sigma–Aldrich, cat. no. 208574).

• Triethylamine4. >99.5%,CRITICAL (C2H5)3N; STEP Sigma–Aldrich,Thermal driftcat. no. may 471283) occur during CAUTION the force Triethylamine spectroscopy experiment and the is harmful uponuser inhalation will lose and the skin depicted absorption, protein and whileis volatile. recording Wear forceappropriate distance gloves curves. and This can be recognized work under a fumefrom hood. a dramatic Store under drop argon in the an bindingd in the dark probability to avoid and amine no oxidation. more characteristic binding events in the • Milli-Q water (Merckretraction Millipore, curve. Burlington, To readjust MA, the USA). cantilever on the depicted spot, switch back to tapping mode • Citric acid (Sigma–Aldrich,and repeat stepscat. no. 6 251275). and 8 from Section 3.4.1 to find/trace back the probed molecule. Do not chance • Stock of membranethe scanning proteins, parameters.reconstituted Startinto proteoliposomes. again with steps UCP1 1–3. and SecYEG for the examples shown here. 5. To calibrate the cantilever spring constant apply the thermal noise method according to • Ethylenediamine (EDA) derivates of the purine nucleotides (EDA-ATP, EDA-ADP and EDA- AMP) (BioLog, HutterBremen, et Germany, al. [27] at cat. the no. end A0 of72, theA118) experiment. or SecA protein Repeat for thetip coupling. spring constant determination for each • Na2SO4 (Sigma-Aldrich,used tip cat. at leastno. 238597). five times. • (NH4)2HPO6.4 (Sigma–Aldrich,Perform a specificity cat. no. 215996). proof, in addition to the amplitude block experiment. After finding a proper • 2-(N-morpholino)ethanesulfonicscanning area with acid (MES) several (Sigma–Aldrich, recognition spots, cat. no. inject M3671). a surface blocking molecule (~1–10 mM) • Tris (Sigma–Aldrich,and scancat. no. this 252859). area until the recognition spots disappear. Use an excess of blocking molecules • Ethylene glycol-bis((mMβ-aminoethyl range) to guarantee ether)-N,N,N that′,N all′-tetraacetic the surface acid molecules (EGTA) (Sigma–Aldrich, are saturated. Ancat. almost complete loss of no. E3889). the recognition events after addition of the free molecule, while leaving the topography image • HEPES (Sigma–Aldrich, cat. no. H3375). unchanged (Figure2c), will clearly confirm that most of the recognition spots were caused by • HEPES potassium salt (K-HEPES) (Sigma–Aldrich, cat. no. H0527). • Sodium Chloridespecific (NaCl) binding(Sigma–Aldrich, of the tip-linked cat. no. S7653). molecule to the proteins in the planar lipid membrane. Select a

Methods Protoc. 2019, 2, x FOR PEER REVIEW 11 of 17

In the first step of applying this combined technique, the distribution of the UCP1 molecules in the formed lipid bilayer as well as their functionality in binding ATP was revealed. Mostly single UCP1 molecules were visualized as round-shaped structures in the topography image, (Figure 2a), whereas the black dots visible in the corresponding recognition image reveal the existence of ATP binding sites on UCP1. As the overlay image of both channels indicates, almost all UCPs that were detected in the topography image were recognized by the ATP-functionalized tip. The specificity of Methodsthe andrecognition Protoc. 2019 signals, 2, 6 was first proven by a so-called amplitude block experiment (Figure 2b), and11 of 18 further by injection of 4 mM free ATP into the buffer solution to competitively block the ATP binding sites in the UCP1 binding pocket (Figure 2c). By varying the amplitude to extreme low or high values, molecule where the recognition has disappeared and record at least one data set of force distance as explained in Section 3.4.1 (step 5), the recognition spots disappeared to appear back again at a propercurves. chosen You amplitude. should detect The almost latter can no be binding clearly events seen in with Figure binding 2b, middle probabilities panel. After below injection 5%, due of to freethe ATP specific in solution blocking (Figure of the 2c), ligands an almost binding comp pocketlete loss in of the recognition receptor. Additionally,spots was observed, recording while some theforce UCP1 distance topography curves remained on the pureunchanged. lipid membrane Likewise, we should do not also see revealany recognition little to no events binding on pure events, lipidsince membranes. specific interaction This clearly with confirms the tip-linkedthat most of ligand the recognition and the lipids spots iswere not caused expected. by specific The few bindingremaining of tip-linked recognition/unbinding ATP to UCP1 molecules events in can the be planar attributed lipid membrane. to unspecific adhesion.

Figure 2. Proof of concept: quantitative characterization of the binding mechanism between Figure 2. Proof of concept: quantitative characterization of the binding mechanism between Uncoupling Protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP) (adapted from Uncoupling Protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP) (adapted from ReferencesReferences [7, 10[7,10]).]). First First part part of of the the combined combined experiment,experiment, simultaneous simultaneous topography topography and and recognition recognition imagingimaging to to visualize visualize specificspecific interaction interaction between between UCP1 UCP1 and andATP. ATP.(a) Topography, (a) Topography, recognition, recognition, and andoverlay overlay image. image. UCP1 UCP1 molecules molecules imaged imaged with with an anAT ATP-tetheredP-tethered tip. tip. Black Black dots dots in the in thecorresponding corresponding recognition image arise from the decrease of the oscillation upwards peaks as a result from UCP1–ATP binding during recognition. The overlay image shows a superposition of the recognition map of UCP1–ATP binding domains (in pink) and the corresponding topography image. Color scale (green to pink) is 0–8 nm. (b) Specificity proof of UCP1–ATP interaction by amplitude block experiment. High resolution topographical (top row) and ATP recognition (bottom row) images of UCP1 recorded at different amplitudes. (c) Specificity of UCP1–ATP interactions tested by a surface block competition experiment. Topographic and recognition images before blocking of UCP1 (left column) and after UCP1 blocking (right column) by adding free ATP (4 mM) into the solution. (a–c) Solid and dashed circles indicate recognized and unrecognized protein molecules, respectively. Recognition events are shown in black. Image sizes: 600 × 600 nm. Methods and Protoc. 2019, 2, 6 12 of 18

4. Expected Results The described protocol provides a method to simultaneously map and study (single) interaction sites at low lateral density by combining AFM-based recognition imaging and force spectroscopy. The first proof-of-concept was applied to the interaction between UCP1 and ATP [7] for examining the binding and inhibition mechanism of UCP1 and UCP3 by three different types of purine nucleotides (PN): ATP, ADP, and AMP [10]. Moreover, by varying the dynamics of force loading, the binding dynamics was elucidated, and the interaction energy landscape was mapped. As a second example the interaction between SecYEG and SecA was studied. Here we show that this mode is not only useful to extensively study ligand binding on a single molecule level, but also to visualize the orientation of the protein in the membrane [28], as presented here for SecYEG.

4.1. Proof-of-Concept: UCP-PN Interaction In the first step of applying this combined technique, the distribution of the UCP1 molecules in the formed lipid bilayer as well as their functionality in binding ATP was revealed. Mostly single UCP1 molecules were visualized as round-shaped structures in the topography image, (Figure2a), whereas the black dots visible in the corresponding recognition image reveal the existence of ATP binding sites on UCP1. As the overlay image of both channels indicates, almost all UCPs that were detected in the topography image were recognized by the ATP-functionalized tip. The specificity of the recognition signals was first proven by a so-called amplitude block experiment (Figure2b), and further by injection of 4 mM free ATP into the buffer solution to competitively block the ATP binding sites in the UCP1 binding pocket (Figure2c). By varying the amplitude to extreme low or high values, as explained in Section 3.4.1 (step 5), the recognition spots disappeared to appear back again at a proper chosen amplitude. The latter can be clearly seen in Figure2b, middle panel. After injection of free ATP in solution (Figure2c), an almost complete loss of recognition spots was observed, while the UCP1 topography remained unchanged. Likewise, we do not see any recognition events on pure lipid membranes. This clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. After identifying ATP-binding UCP1 molecules, switching from tapping to contact mode and positioning of the cantilever on a selected recognition spot was accomplished. Force spectroscopy experiments were started to probe the binding activity and interaction forces at different pulling speeds. Depending on the applied pulling speed, the binding frequency ranged between 30 and 45% and decreased dramatically to below 5% after injecting a blocking molecule (mM range, excess to guarantee a full saturation of UCP1 molecules) or probing the interaction on a pure lipid membrane lacking the UCP1 molecule. After analyzing the FDCs recorded on UCP1 proteins with respect to unbinding force, loading rate and effective spring constant, a dynamic force spectroscopy (DFS) plot from all the applied pulling speeds was reconstructed. After fitting the data force vs. loading rate data points with the Bell–Evans model [29] (Figure3a), two important parameters of the interaction energy landscape of UCP1-ATP binding were extracted: the characteristic length scale of the energy barrier, −1 xβ ~ 1.65 ± 0.01 Å, and the kinetic off-rate, koff = 0.89 ± 0.11 s , which is the inverse of the bond lifetime, τ = 1.12 ± 0.14 s. After the successful proof-of-concept, we further applied this method to study the binding and inhibition of UCP3 (another uncoupling protein) in comparison to UCP1 by different purine nucleotides (Figure3b). Both proteins are mitochondrial membrane proteins, expressed in different tissues (e.g., brown adipose tissue—BAT), and facilitate proton leakage to support non-shivering thermogenesis. For UCP3, the transport function and the molecular mechanism of the regulation are poorly investigated. Interestingly our data show that the lifetimes of both UCP3-PN (1.33 s, 0.56 s, and 0.13 s for ATP, ADP, and AMP, respectively) and UCP1-PN (1.12 s, 0.75 s, and 0.16 s for ATP, ADP, and AMP, respectively) interactions decrease alongside with the degree of PN-phosphorylation (Figure3b) [10]. Methods Protoc. 2019, 2, x FOR PEER REVIEW 12 of 17

recognition image arise from the decrease of the oscillation upwards peaks as a result from UCP1– ATP binding during recognition. The overlay image shows a superposition of the recognition map of UCP1–ATP binding domains (in pink) and the corresponding topography image. Color scale (green to pink) is 0–8 nm. (b) Specificity proof of UCP1–ATP interaction by amplitude block experiment. High resolution topographical (top row) and ATP recognition (bottom row) images of UCP1 recorded at different amplitudes. (c) Specificity of UCP1–ATP interactions tested by a surface block competition experiment. Topographic and recognition images before blocking of UCP1 (left column) and after UCP1 blocking (right column) by adding free ATP (4 mM) into the solution. (a–c) Solid and dashed circles indicate recognized and unrecognized protein molecules, respectively. Recognition events are shown in black. Image sizes: 600 × 600 nm.

After identifying ATP-binding UCP1 molecules, switching from tapping to contact mode and positioning of the cantilever on a selected recognition spot was accomplished. Force spectroscopy experiments were started to probe the binding activity and interaction forces at different pulling speeds. Depending on the applied pulling speed, the binding frequency ranged between 30 and 45% and decreased dramatically to below 5% after injecting a blocking molecule (mM range, excess to guarantee a full saturation of UCP1 molecules) or probing the interaction on a pure lipid membrane lacking the UCP1 molecule. After analyzing the FDCs recorded on UCP1 proteins with respect to unbinding force, loading rate and effective spring constant, a dynamic force spectroscopy (DFS) plot from all the applied pulling speeds was reconstructed. After fitting the data force vs. loading rate data points with the Bell–Evans model [29] (Figure 3a), two important parameters of the interaction energy landscape of UCP1-ATP binding were extracted: the characteristic length scale of the energy −1 Methodsbarrier, and Protoc. xβ 2019~ 1.65, 2 ,± 6 0.01 Å, and the kinetic off-rate, koff = 0.89 ± 0.11 s , which is the inverse of the bond13of 18 lifetime, τ = 1.12 ± 0.14 s.

Methods Protoc. 2019, 2, x FOR PEER REVIEW 13 of 17

tissues (e.g., brown adipose tissue—BAT), and facilitate proton leakage to support non-shivering thermogenesis. For UCP3, the transport function and the molecular mechanism of the regulation are poorly investigated. Interestingly our data show that the lifetimes of both UCP3-PN (1.33 s, 0.56 s, and 0.13 s for ATP, ADP, and AMP, respectively) and UCP1-PN (1.12 s, 0.75 s, and 0.16 s for ATP, ADP, and AMP, respectively) interactions decrease alongside with the degree of PN-phosphorylation (Figure 3b) [10].

4.2. SecYEG Figure 3. Proof of concept: quantitative characterization of the binding mechanism between UCP1 and VisualizationFigure 3. Proof of SecYEGof concept: molecules quantitative on characteri the samplezation surface of the binding was achieved mechanism by between fusion UCP1of SecYEG its inhibitorand its ATP inhibitor (adapted ATP from(adapted References from References [7,10]). Dynamic[7,10]). Dynamic force spectroscopy force spectroscopy to study to study the binding the proteoliposomes on freshly cleaved mica resulting in formation of a planar lipid bilayer. forces betweenbinding forces a pre-selected between a UCPpre-selected molecule UCP and molecule its corresponding and its corresponding purine nucleotide purine nucleotide (ATP, ADP, (ATP, AMP). Topographical AFM images showed a low SecYEG density on the sample surface, making the (a) DynamicADP, AMP). force spectroscopy(a) Dynamic force plot spectroscopy for the UCP1–ATP plot for interaction.the UCP1–ATP Single-binding interaction. Single-binding events between acquisition of accurate and reliable force spectroscopy experiments difficult. By measuring the size UCP1events and ATP between are shown UCP1 and as green ATP are dots. shown After as applyinggreen dots. the After Bell–Evans applying the model Bell–Evans (black model line), (black the bond of SecYEG molecules on the topographical images, an average height of ~1.2 nm was determined lifetimeline), can the be bond directly lifetime determined. can be directly (b) Application determined. of(b) this Application combined of this mode combined to other mode UCP-PN to other pairs: (Figure 4c). This value is in a good agreement with the values reported in the literature [30] for the DependenceUCP-PN of pairs: bond Dependence lifetimes onof bond PN-phosphorylation lifetimes on PN-phosphorylation for UCP1-PN for (blue) UCP1-PN and (blue) UCP3-PN and UCP3- (green). protrusionPN of(green). the cytoplasmic Shown are the site mean of SevaluescYEG ± SD out of ofat leastthe membraneindependent (Figuremeasurements, 4e). arising from the Shown are the mean values ± SD of at least independent measurements, arising from the fit in (a). Detectionfit in (a). and localization of functional SecYEG molecules at such a low surface density was 4.2.achieved SecYEG by employing AFM-based force spectroscopy guided by recognition imaging. An acousticallyAfter oscillating the successful AFM proof-of-concept, tip carrying covalent we furtherly attached applied thisSecA method was scanned to study overthe binding the sample and surfaceVisualizationinhibition (Figure of 4a). ofUCP3 SecYEGSpecific (another binding molecules uncoupling of SecA on the toprotein) surface-immobilized sample in surfacecomparison was SecYEGto achieved UCP1 resulted by by different fusion in generation ofpurine SecYEG proteoliposomesof anucleotides recognition on(Figure signal freshly 3b). (dark cleaved Both spots proteins mica in resultingFigure are mitoch 4b in).ondrial Mapping formation membrane SecA of a planarbinding proteins, lipid sites expressed bilayer. by TREC Topographicalin different allowed AFMtargeting images of showed functional a low SecYEG SecYEG channels density with on the nanometer sample surface, accuracy making for force the acquisitionmeasurements. of accurate In a andsimilar reliable manner force spectroscopyas thoroughly experiments described in difficult. Section 4.1, By measuringby switching the to size force of SecYEGspectroscopy molecules mode on the(Figure topographical 4d), binding images, probabilities, an average unbinding height of forces, ~1.2 nm and was information determined on the (Figure energy4c). landscape This value of is the in a goodinteraction agreement between with thethevalues ATP motor-protein reported in the SecA literature and the [30 ]translocation for the protrusion channel of theSecYEG cytoplasmic were siteextracted. of SecYEG out of the membrane (Figure4e).

FigureFigure 4. 4.Proof Proof of of concept: concept: characterization characterization ofof thethe SecA–SecYEGSecA–SecYEG interaction. interaction. (a ()a Scheme) Scheme of ofSecYEG SecYEG complexescomplexes reconstituted reconstituted into into a planara planar lipid lipid bilayer bilayer with with the the tip tip carrying carrying a a SecA SecA molecule molecule attached attached viavia an N-hydroxysuccinimidean N-hydroxysuccinimide (NHS)–PEG–Acetal (NHS)–PEG–Acetal linker. linker. (b) SecYEG(b) SecYEG molecules molecules that that interacted interacted with with the the SecA onSecA the tip on appear the tip as appear dark spots as dark in the spots recognition in the recognition images. (c) images. Cross-section (c) Cross-section of SecYEG. (ofd )SecYEG. Characteristic (d) force-distanceCharacteristic curve force-distance for the SecYEG–SecA curve for the interaction. SecYEG–SecA (e) Side interaction. view of the(e) crystalSide view structure of the of crystal SecYEG fromstructureM. Jannaschii of SecYEG(PDB-ID from M. 1RHZ). Jannaschii (PDB-ID 1RHZ).

4.3. Data Analysis This step depends on the AFM type and producer used; data analysis tools are usually supplied along with the AFM. Two main types of data analysis need to be carried out. First, the analysis of the AFM topography and recognition images, followed by the analysis of the force curves acquired. In the following, some basic guidelines to analyze and interpret the data are given.

Methods and Protoc. 2019, 2, 6 14 of 18

Detection and localization of functional SecYEG molecules at such a low surface density was Methods Protoc. 2019, 2, x FOR PEER REVIEW 4 of 17 achieved by employing AFM-based force spectroscopy guided by recognition imaging. An acoustically oscillating AFM tip carrying covalently attached SecA wascharacterization scanned over of the the sample binding surface mechanism (Figure between4a). mitochondrial membrane uncoupling protein 1 Specific binding of SecA to surface-immobilized SecYEG(UCP1) resulted and in its generation inhibitor adenosine of a recognition triphosphate signal (ATP). Here the binding dynamics as well as the (dark spots in Figure4b). Mapping SecA binding sitesinteraction by TREC energy allowed landscape targeting was of elucidated functional [7,14] (Section 4.1). In the second example, the SecYEG channels with nanometer accuracy for force measurements.interaction between In a similar the bacterial manner translocation as thoroughly channel SecYEG and the cytoplasmic motor-protein described in Section 4.1, by switching to force spectroscopySecA modewas characterized. (Figure4d), bindingThe AFM-based probabilities, force spectroscopy guided by recognition imaging was employed in order to localize the SecYEG complexes and map SecA binding sites on suspended lipid unbinding forces, and information on the energy landscape of the interaction between the ATP bilayers. Subsequently, the interaction forces between SecYEG and SecA interaction were motor-protein SecA and the translocation channel SecYEG were extracted. characterized. Finally, Section 4.3 provides detailed information on how the data were analyzed and interpreted. 4.3. Data Analysis This step depends on the AFM type and producer used;2.1. Materials data analysis tools are usually supplied along with the AFM. Two main types of data analysis need• toAcetal–PEG be carried27–NHS out. First, (available the analysis by purchase of the from H. Gruber, Johannes Kepler University Linz AFM topography and recognition images, followed by the analysis[15], Linz, of theAustria). force Alternatively, curves acquired. this linker In the can be synthesized according to Reference [16]. following, some basic guidelines to analyze and interpret theCRITICAL data are given.STEP We recommend purchasing monodisperse polyethylene glycol (PEG) linkers from this source for two reasons. First, the benzaldehyde function is protected by an acetal- 4.3.1. AFM Topography and Recognition Images group for the binding to amino-functionalized tips via the activated carboxyl functionality of the N-hydroxysuccinimide (NHS)-group. This elegantly precludes the possibility of bivalent 1. Load the topography and recognition image into Gwyddion and visualize the in a false-color interaction of the benzaldehyde function with adjacent amino-groups on the tip surface. Second, image (topography: e.g., “golden”; recognition: e.g., “greythe linker scale” is supplied or any otherin high-purity color pattern 1-mg aliquots to ideally suited for use in this protocol. properly distinguish between topography and adhesion• Dimethylsulfoxide events). anhydrous (>99.9%, (CH3)2SO (DMSO); Sigma–Aldrich, St. Louis, MO, USA, 2. Improve the height images by using filters such as flattencat. or no. plane 41640) fit. CRITICALCAUTION DMSO STEP Usersis harmful upon inhalation and skin absorption. Wear should keep in mind that application of any filter will—toappropriate some extent—modify gloves. the raw values • and that quantitative parameters should be extracted beforeSodium use cyanoborohydride of filters. (NaCNBH3; Sigma–Aldrich, cat. no. 156159) CAUTION NaCNBH3 is highly toxic. Wear gloves and handle carefully. 3. In order to analyze the height and diameter of the (single) molecules, use a zoom-in image and • Chloroform (>99.9%, CHCl3; Sigma–Aldrich, cat. no. 288306) CAUTION Chloroform is extract cross-sectional profiles. highly volatile and harmful upon inhalation and skin absorption. Wear appropriate gloves and 4. For allocation of recognition events to topographicalwork features, under producea fume hood. an Handle overlay only of thosein glass vessels and glass dishes covered with a lid. two channels. • Ethanol, absolute (>99.9%, C2H5OH; Sigma–Aldrich, cat. no. 24102). 5. For comparing different amplitudes at the same scan• area(3-Aminopropyl)triethoxysilane (amplitude block) or the images (APTES, taken 99%, Sigma–Aldrich, cat. no. 440140). Distill at low before/after the surface block, keep the z-scale valuetemperature (to adjust and the contrast),store under as argon well in as se thealed crimp vials over silica gel at −20 °C to avoid threshold (recognition yes or no) always constant. Determinepolymerization. the right threshold from regions • without a protein; only when the recognition signal is belowEthanolamine this value, hydrochloride consider the (H protein2NC2H4OH; as to Sigma–Aldrich, cat. no. E6133). • 0.1 mM NaOH solution (in water, Sigma–Aldrich, cat. no. 43617). be recognized by the ligand functionalized tip. • Molecular sieves, 3 Å (beads, 8–12 mesh; Sigma–Aldrich, cat. no. 208574). • 4.3.2. Analysis of the Force Curves Triethylamine >99.5%, (C2H5)3N; Sigma–Aldrich, cat. no. 471283) CAUTION Triethylamine is harmful upon inhalation and skin absorption, and is volatile. Wear appropriate gloves and For the determination of interaction forces between a specificwork receptor—ligandunder a fume hood. pair, Store force-distance under argon and in the dark to avoid amine oxidation. cycles are recorded. The cantilever behaves like a Hookeian• Milli-Q spring, water where (Merck the forceMillipore, F exerted Burlington, on the MA, USA). sample by the AFM tip scales linearly with the cantilever• deflectionCitric acid (Sigma–Aldrich,∆z, according tocat. Hooke’s no. 251275). law: • F = kc∆z, kc being the cantilever’s spring constant and z the defection,Stock of membrane which should proteins, be proportional reconstituted to into proteoliposomes. UCP1 and SecYEG for the the travel distance of the z-piezo in the contact region of the force-distanceexamples shown cycle. here. The retraction segment • Ethylenediamine (EDA) derivates of the purine nucleotides (EDA-ATP, EDA-ADP and EDA- of the FD curves is further used for data analysis of unbinding events. To assess the specificity of the AMP) (BioLog, Bremen, Germany, cat. no. A072, A118) or SecA protein for tip coupling. interaction, adhesion events must be separated from the contact region of the FD curve. Practically, • Na2SO4 (Sigma-Aldrich, cat. no. 238597). the rupture distance of a specific adhesion event will depend on the extended length of the PEG • (NH4)2HPO4 (Sigma–Aldrich, cat. no. 215996). linker at the applied force, the size of the molecule attached• 2-(N-morpholino)ethanesulfonic to the PEG linker and the probed acid sample. (MES) (Sigma–Aldrich, cat. no. M3671). Moreover, by utilizing appropriate biophysical models, the• kineticTris (Sigma–Aldrich, and thermodynamic cat. no. 252859). parameters of the specific binding events can be extracted to give insight• intoEthylene the binding glycol-bis( free-energyβ-aminoethyl landscape. ether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma–Aldrich, cat. no. E3889). 1. Load the recorded FD-curves into suitable scientific• HEPES analysis (Sigma–Aldrich, software, cat. such no. H3375). as Matlab (custom-written routines from Keysight commercially• available),HEPES potassium Gwyddion salt (K-HEPES) or PicoView. (Sigma–Aldrich, cat. no. H0527). • 2. Depending on the used software, extract the following Sodium values: Chloride (NaCl) (Sigma–Aldrich, cat. no. S7653).

Methods and Protoc. 2019, 2, 6 15 of 18

a. Loading rate from each rupture event: Measure the slope of the force versus distance curve just before the rupture. This gives the value of the effective spring constant keff (force segment ∆F divided through the distance segment ∆x of the very last part of the rupture event). Multiply this value with the applied pulling speed. Alternatively, as a more elegant way: If force vs. time curves were additionally recorded, the slope of the force vs. time curve just before the rupture event directly results in the loading rate. b. Rupture force: Take the value on the y-axis (force axis) right on the (negative) maximum peak of the rupture event before it returns to baseline level. In case of curves containing multiple rupture events, each rupture should be considered separately. c. Plot the extracted unbinding force versus the loading rate on a semi-logarithmic scale (DFS plot) using Origin or a similar software, which allows to extract kinetic and thermodynamic parameters using appropriate models and strategies [31]. d. If desired, make unbinding force histograms or probability density functions (pdfs) from separated, distinct loading rate ranges to capture all the information contained in the DFS plot, such as the presence of multiple interactions. Fit the histograms with Gaussian distributions to determine the presence of single or multiple peaks, directly corresponding to one or several receptor–ligand interaction(s). The advantage of using pdfs instead of histograms is that data binning is avoided and the data will be analyzed more precisely, since the values are weighted to their reliability [32]. e. Fit the loading-rate-dependent interaction forces with the fitting model of choice. Such models can be a linear iterative fitting algorithm (Levenberg Marquardt) along with the Bell–Evans model [29] (Figure3a) or a non-linear iterative fitting algorithm (Levenberg Marquardt) along with the Friddle–Noy–de-Yoreo (FNdY) model [33]. The simplest and most commonly used model is the Bell–Evans fit for crossing a single energy barrier, where the user is obliged just to take single interactions into account. Here, exclude multiple binding events from the force pdfs, which appear as a “shoulder” in the Gaussian distribution of the most probable unbinding force. Exclude these data and use only data within the interval [µ ± σ] for construction of the DFS plots and the subsequent fitting with the Bell–Evans model. f. An extensive overview of analysis and fitting of dynamic force spectroscopy (DFS) data is given by Bizzarri and Cannistraro [1], as well as by Noy and Friddle [33], whereas Hane and colleagues have compared the dominant models in great detail [34]. The choice of model for data analysis relies first and foremost on the obtained force spectrum; non-linear alternatives to the Bell–Evans model should be used only if the forces measured do not scale linearly with the logarithm of the loading rate—as fitting a linear dependence with a non-linear function may result in fit parameters with little to no meaning.

5. Reagents Setup • Assay buffer.

UCP1–ATP example: 50 mM NaSO4, 10 mM MES, 10 mM Tris, 0.6 mM EDTA, adjusted to # pH 7.2.

SecYEG–SecA example: 50 mM Tris, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, adjusted to # pH 7.0. ◦ • Citric acid 1% (w/v). Dissolve 0.5 g of citric acid in 50 mL of Milli-Q H2O. Store at −20 C in 2-mL aliquots for up to 2 years.

• Ethanolamine solution (1 M, pH 8.0). Dissolve 1.2 g of ethanolamine in 20 mL of Milli-Q H2O. Adjust the pH to 8.0 with 1 M HCl. Store at −25 ◦C in 0.5-mL aliquots for up to 2 years. Methods Protoc. 2019, 2, x FOR PEER REVIEW 4 of 17

characterization of the binding mechanism between mitochondrial membrane uncoupling protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP). Here the binding dynamics as well as the interaction energy landscape was elucidated [7,14] (Section 4.1). In the second example, the interaction between the bacterial translocation channel SecYEG and the cytoplasmic motor-protein SecA was characterized. The AFM-based force spectroscopy guided by recognition imaging was employed in order to localize the SecYEG complexes and map SecA binding sites on suspended lipid bilayers. Subsequently, the interaction forces between SecYEG and SecA interaction were characterized. Finally, Section 4.3 provides detailed information on how the data were analyzed and interpreted.

2.1. Materials Methods and Protoc. 2019, 2, 6 16 of 18 • Acetal–PEG27–NHS (available by purchase from H. Gruber, Johannes Kepler University Linz [15], Linz, Austria). Alternatively, this linker can be synthesized according to Reference [16]. • Sodium cyanoborohydrideCRITICAL (1 M solutionSTEP We withrecommend 20 mM purchasing NaOH). Working monodisperse under polyethylene a chemical glycol hood, (PEG) linkers from this source for two reasons. First, the benzaldehyde function is protected by an acetal- place 13 mg of NaCNBH3 into a polyethylene capped glass weighing bottle. Pipette 20 µL of group for the binding to amino-functionalized tips via the activated carboxyl functionality of the 100 mM NaOH on top of the NaCNBH and mix well by pipetting. Then add 180 µL of Milli-Q N-hydroxysuccinimide3 (NHS)-group. This elegantly precludes the possibility of bivalent water and mix by pipetting.interaction Ifthe of the balance benzaldehyde used for function weighing with isadja outsidecent amino-groups of the chemical on the hood, tip surface. Second, first zero the balancethe using linker the is emptysupplied weighing in high-purity bottle 1-mg with aliquots lid on, ideally and suited then addfor use ~13 in mgthis protocol. of NaCNBH3 to the• bottle Dimethylsulfoxide under the hood. anhydrous Cap the (>99.9%, bottle and (CH weigh.3)2SO (DMSO); Adjust Sigma–Aldrich, volumes of NaOH St. Louis, MO, USA,

and Milli-Q water proportionately.cat. no. 41640) CAUTIONCAUTION DMSONaCNBH is harmful3 is highly upon toxic. inhalation Wear and gloves skin andabsorption. Wear handle with care. Handleappropriate under gloves. a chemical hood during preparation. CAUTION Prepare • Sodium cyanoborohydride (NaCNBH3; Sigma–Aldrich, cat. no. 156159) CAUTION fresh NaCNBH3 for each functionalization. Dispose of any remaining NaCNBH3 according to NaCNBH3 is highly toxic. Wear gloves and handle carefully. institutional regulations. • Chloroform (>99.9%, CHCl3; Sigma–Aldrich, cat. no. 288306) CAUTION Chloroform is highly volatile and harmful upon inhalation and skin absorption. Wear appropriate gloves and Author Contributions: Conceptualization, M.K. and P.H.; Methodology, M.K. and P.H.; Validation, M.K. and work under a fume hood. Handle only in glass vessels and glass dishes covered with a lid. P.H.; Formal Analysis, M.K.;• Investigation, M.K. and A.F.; Resources, M.K., A.F., H.J.G. and P.H.; Data Curation, M.K. and A.F.; Writing—Original Ethanol, Draft absolute Preparation, (>99.9%, M.K. C2 andH5OH; A.F.; Sigma–Aldrich, Writing—Review cat. &no. Editing, 24102). M.K., A.F. and P.H.; Visualization, M.K. and• A.F.;(3-Aminopropyl)triethoxysilane Supervision, P.H.; Project Administration, (APTES, 99%, P.H.; Sigma–Aldrich, Funding Acquisition, cat. no. 440140). P.H. Distill at low temperature and store under argon in sealed crimp vials over silica gel at −20 °C to avoid Funding: This research was funded by Austrian Research Fund (FWF) grant number P 25357 (M.K.) and grant number W1250-B200 (A.F.). polymerization. • Ethanolamine hydrochloride (H2NC2H4OH; Sigma–Aldrich, cat. no. E6133). Acknowledgments: We thank the laboratory of Elena E. Pohl (Institute of Physiology, Pathophysiology and • 0.1 mM NaOH solution (in water, Sigma–Aldrich, cat. no. 43617). Biophysics, University of Veterinary• Medicine, Vienna, Austria), in particular Gabriel Macher and Anne Rupprecht, for kindly providing the studied Molecular membrane sieves, protein 3 Å UCP. (beads, Moreover, 8–12 mesh; we thank Sigma–Aldrich, the laboratory cat. of no. Peter 208574). Pohl (Institute of Biophysics, Johannes Kepler• Triethylamine University, Linz, >99.5%, Austria), (C2H in5)3 particularN; Sigma–Aldrich, Denis Knyazev, cat. no. Mirjam 471283) Zimmermann CAUTION and Triethylamine Roland Kuttner, for kindly providingis harmful the upon studied inhalation proteins and SecYEG skin and absorption, SecA. and is volatile. Wear appropriate gloves and Conflicts of Interest: The authorswork declare under noa fume conflict hood. of interest.Store under argon and in the dark to avoid amine oxidation. • Milli-Q water (Merck Millipore, Burlington, MA, USA). • References Citric acid (Sigma–Aldrich, cat. no. 251275). • Stock of membrane proteins, reconstituted into proteoliposomes. UCP1 and SecYEG for the 1. Bizzarri, A.R.; Cannistraro,examples S. The shown application here. of atomic force spectroscopy to the study of biological complexes undergoing• aEthylenediamine biorecognition process. (EDA)Chem. derivates Soc. of Rev. the2010 purine, 39, 734–749.nucleotides [CrossRef (EDA-ATP,][PubMed EDA-ADP] and EDA- 2. Cleveland, J.; Radmacher,AMP) M.; (BioLog, Hansma, Bremen, P. Atomic Germany, Scale Force cat. Mappingno. A072, withA118) the or Atomic SecA protein Force Microscope for tip coupling.; • Springer: Dordrecht, TheNa Netherlands,2SO4 (Sigma-Aldrich, 1995. cat. no. 238597). • 3. Heinz, W.F.; Hoh, J.H. Spatially(NH4)2HPO resolved4 (Sigma–Aldrich, force spectroscopy cat. no. of215996). biological surfaces using the atomic force • microscope. Trends Biotechnol. 2-(N-morpholino)ethanesulfonic1999, 17, 143–150. [CrossRef acid] (MES) (Sigma–Aldrich, cat. no. M3671). • 4. Ludwig, M.; Dettmann, Tris W.; (Sigma–Aldrich, Gaub, H. Atomic cat. force no. 252859). microscope imaging contrast based on molecular • Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma–Aldrich, cat. recognition. Biophys. J. 1997, 72, 445–448. [CrossRef] no. E3889). 5. Rico, F.; Su, C.; Scheuring, S. Mechanical mapping of single membrane proteins at submolecular resolution. • HEPES (Sigma–Aldrich, cat. no. H3375). Nano Lett. 2011, 11, 3983–3986. [CrossRef][PubMed] • HEPES potassium salt (K-HEPES) (Sigma–Aldrich, cat. no. H0527). 6. Heu, C.; Berquand, A.; Elie-Caille, C.; Nicod, L. Glyphosate-induced stiffening of hacat keratinocytes, a peak • Sodium Chloride (NaCl) (Sigma–Aldrich, cat. no. S7653). force tapping study on living cells. J. Struct. Biol. 2012, 178, 1–7. [CrossRef]

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