This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.
In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.
Beside optical and magnetic tweezers, the atomic force microscope (AFM)1,2 has emerged as a useful tool to analyze and manipulate molecules and probe their properties and functions, including their response to external force3,4. In contrast to methods like the enzyme linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or quartz crystal microbalance (QCM) setups, AFM allows to measure interactions on the single molecule (SMFS)5 and single cell level (SCFS)6. These technologies yielded valuable insight into binding mechanisms like the catch bonds found for the interaction of E. coli pilus protein FimH with mannose7, or the tandem β-zipper repeats formed by Fn binding proteins from S. aureus upon binding to Fn8. We were recently able to show that the pilus-1 adhesin RrgA9,10 from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae)11 is able to bind to fibronectin12 with its two terminal domains. This revealed a new two-domain binding mechanism which differs from the tandem β-zipper and may enable piliated pneumococci to form and maintain a transient contact to fibronectin-containing host surfaces13.
The success of SMFS experiments critically depends on the functional and native immobilization of the biomolecules on solid surfaces and AFM tips. As high forces may occur during SMFS measurements, the proteins should preferably be covalently coupled to the surface. There are a large number of different coupling methods for the immobilization of proteins and other biomolecules, as well as whole cells on (inorganic) solid surfaces, nano- particles and other devices described in the literature14,15,16,17,18,19,20,21,22,23,24,25,26,27. These protocols often make use of hazardous substances, are difficult to perform and/or require special equipment (e.g., plasma cleaner). A simple way to couple molecules to glass is to attach a thicker polymer layer of heterobifunctional crosslinkers with a silane-reactive group on one side and an amine-reactive group on their other side. Depending on the application, the coupling agents can comprise flexible hydro-carbon chains of variable length, e.g., polyethylenglycol (PEG). They suppress non-specific interactions of the modified surfaces (e.g., hydrophobic, electrostatic and van-der-Waals interactions) and may provide the coupled molecule rotational freedom.
Here, we describe a general protocol for the covalent coupling of proteins containing one or more free amino groups (-NH2) to glass surfaces and silicon nitride AFM tips via a heterobifunctional ethoxy silane-PEG-carboxyl (-COOH). This protocol can be used in SMFS experiments, which is exemplified based on the interaction of RrgA and the extracellular matrix protein Fn (see Figure 1 for an overview).
The first step is the silanization of the surface28,29,30,31. It involves the hydrolysis of the ethoxy groups of the coupling agent in order to form highly reactive SiOH groups. These can react with SiOH groups on the substrate. In a primary condensation step, these silanols form hydrogen bonds and spread on the substrate. In a secondary condensation reaction (which usually requires heat or vacuum to remove water), siloxane bonds are formed. This results in a covalently attached organo-silane layer.
The second step is the coupling of the proteins to the functional (-COOH) groups which extend from the polymer32. First, the acid is converted to a reactive N-hydroxysuccinimid (NHS) ester intermediate, which is gained through the well-established NHS/EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimid chemistry33 and undergoes nucleophilic substitution to finally form an amide bond with primary amines on the proteins.
In this way, RrgA was coupled to silicon nitride AFM tips and human Fn to glass substrates in a random orientation and their interaction forces were analyzed on the single molecule level. Our results show that the described surface chemistry leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the tip, apparent by the target value of up to 20% of interaction events during SMFS measurements. This chemistry reduces non-specific background interactions, is subject to little alteration during data acquisition and is therefore excellently suited for precise SMFS experiments.
1. Immobilization of Proteins via Functional Silane Coupling Agents
Note: Figure 1 gives an overview over the surface chemistry applied in this protocol.
CAUTION: In the following protocol, different chemicals with corrosive and skin irritating properties are used. Wear adequate (acid-resistant) gloves, safety goggles, and laboratory coat and work under the fume hood while preparing solutions in order to avoid inhalation of vapors.
2. Atomic Force Microscopy Based Single Molecule Force Spectroscopy
Note: In this work, an atomic force microscope from JPK Instruments was used and the set ups for obtaining force-distance curves was defined with the Force RampDesigner.
The protocol described here results in a covalent immobilization of proteins via their accessible primary amines with random orientation (Figure 1). Figure 2 shows an AFM image of a silanized glass surface with (left) and without (right) Fn immobilized, recorded after the dehydration of the samples under a gentle stream of nitrogen. The silane polymer layer shows only small surface corrugations with a height of approximately 2-5 nm (Figure 2, right), while on the surface functionalized with Fn, approximately 10 nm high Fn molecules are apparent (Figure 2, left). In close-ups, the dimeric structure of the Fn can be recognized. The Fn molecules seem to be compact with a height of 4 – 5 nm above the PEG surface coating and a length of ~ 120 nm (see inserts).
To investigate the interaction forces of RrgA with Fn, which was recently described in detail by our group13, RrgA was coupled to a silicon nitride AFM tip and human Fn to the glass substrate (Figure 3a). Figure 3 shows representative tip sample separation curves of the interaction of RrgA with Fn recorded at a pulling velocity of 1 µm s-1. The used surface chemistry led to a low background interaction and well-shaped single (or double) interaction events (Figure 3a), which were fitted using an extensible worm like chain (eWLC) model (red curves). Plotting the results of the fit (rupture force and -length, see Figure 4) shows that after overcoming non-specific surface interactions between AFM tip and substrate, and stretching the PEG linkers (> 70 nm), up to ~19% of the force curves showed rupture events with a mean rupture force for the RrgA – Fn interaction of ~ 52 pN at a tip-sample distances of about 100 nm. In contrast, an inapplicable surface chemistry (here, omitted quenching with Tris buffered saline Figure 3b) will hinder a clear evaluation of single interaction events due to unspecific interactions, multiple protein binding (trace 2 and 3) and/or covalent coupling of proteins between the sample surface and the AFM cantilever tip. This leads to high rupture forces (trace 1) possibly accompanied by unfolding of the protein (Fn) domains (trace 4 and 5).
Figure 1: Overview over the surface chemistry. The hydrolysis of ethoxy silane-PEG-carboxyl is followed by its condensation at the hydrated glass surface and the formation of siloxane crosslinks. The reaction of EDC with the carboxyl groups results in a reactive o-acylisourea, an amine-reactive intermediate with an extremely short half-life in aqueous solution (hydrolysis). The intermediate is stabilized by the formation of an NHS ester which undergoes nucleophilic substitution to finally form an amide bond with primary amines on the proteins. Please click here to view a larger version of this figure.
Figure 2: Immobilization of fibronectin on a glass substrate via heterobifunctional ethoxy silane PEG carboxyl coupling agent. AFM images of functionalized glass surfaces with (left) and without (right) Fn. Numbers indicate individual Fn molecules, which are distributed homogeneously on the substrate surface (inserts). The molecules adopt a dimeric and compact structure with a height of 4-5 nm above the PEG coating and a length of >100 nm. This resembles the structure of Fn in solution and is consistent with previous AFM data on other surfaces, e.g., mica (scale bar of inlays = 500 nm)37. Below the AFM images are height profiles along the lines indicated in the AFM images. Please click here to view a larger version of this figure.
Figure 3: Illustration of a SMFS experiment and representative force distance curves of the RrgA – Fn interaction. (a) RrgA and Fn were covalently linked via the heterobifunctional ethoxy silane PEG carboxyl coupling agent to a silicon nitride AFM cantilever tip and a glass surface, respectively. Representative SMFS force distance curves obtained for RrgA – Fn interaction at a retraction velocity of 1 µm s-1 with the described immobilization of RrgA and Fn are shown. Red curves represent the extensible worm-like chain fits applied to obtain rupture forces and lengths. The figure has been modified from Becke, et al., ACSnano 201813. (b) Representative SMFS force distance curves obtained for the RrgA – Fn interaction without quenching with Tris buffered saline. In this case, the primary amine of Tris was absent so that remaining active NHS esters were left unsaturated during the experiment. This led to multi protein binding (trace 2 and 3) and the clamping of proteins between the surface and the AFM tip which resulted in high rupture forces accompanied by (Fn-) domain unfolding (trace 1, 4 and 5; note the different scales). Please click here to view a larger version of this figure.
Figure 4: Force and length distribution of single RrgA – Fn interactions. Rupture force and corresponding rupture length histograms obtained from RrgA – Fn SMFS interaction measurements (n = 1400) at a retraction velocity of 1 µm s-1. The histograms reveal a most probable rupture force fMP of 51.6 pN (Gauss fit, black line) and an accumulation of rupture lengths around 100 nm. The figure has been modified from Becke, et al., ACSnano, 201813. Please click here to view a larger version of this figure.
Since the introduction of AFM based SMFS, it evolved into a widely used technique to directly probe intra and intermolecular forces of individual proteins, nucleic acids and other biomolecules3,4,5. For successful SMFS experiments, an appropriate surface coupling strategy is a prerequisite. To probe the intramolecular forces in natural and synthetic polymers, the polymers may be directly coupled to the substrate surface and AFM tip36,38,39,40,41. For the investigation of inter-molecular interactions, such as molecular bonds, however, it is advisable to use flexible linker molecules such as hetero-bifunctional PEG linkers, or polypeptide chains, to attach the interaction partners to the AFM tip and the substrate surface, in order to allow for the correct orientation of the binding partners, to overcome short-range surface forces and to avoid denaturation and unfolding of proteins21,22,23,24,25,26,27,42. We therefore described a simple and straight forward protocol for the covalent immobilization of proteins via their accessible primary amines using hetero-bifunctional PEG spacers.
We demonstrated its applicability with the investigation of the interaction forces between adhesin RrgA from S. pneumoniae and the extracellular matrix protein Fn, as recently described in detail elsewhere13.
The surface chemistry is well established and analyzed and similar approaches have been successfully used in multiple SMFS experiments19,42,43,44,45. The silylether used for coupling the silane polymer to the surface, is subject to hydrolysis. The degree of hydrolysis depends on the amount of formed siloxane bonds, which can be controlled during the silanization process. If high interaction forces (≥ 1000 pN) are expected during SMFS measurements, the silanization should be performed via vapor-phase deposition30 which results in the formation of a continuous layer of siloxanes. As for many experiments (e.g., many protein – protein interactions), the interaction forces are in the range of a few hundred pN, and the described procedure, in which siloxane formation is carried out by deposition from an aqueous phase and unbound organo-silanes are thoughtfully washed off with ethanol (step 1.1.7) followed by curing with heat (step 1.1.8), is sufficient.
Another critical step is to wash the remaining EDC and NHS molecules off the surface (step 1.2.3), as leftovers will lead to the activation of carboxyl groups on the proteins. This may either result in crosslinking of proteins on the same surface, which can alter their functionality or covalently couple activated proteins to other proteins on the opposite surface. This may lead to clamping of the proteins between the surface and the AFM tip, which results in high rupture forces possibly accompanied by domain unfolding (see Figure 3b, trace 1, 4 and 5, unfolding of Fn domains)46. The same problem can occur, if the active NHS esters of the PEG spacer are left unsaturated. Therefore, the incubation with Tris buffered saline is recommended (step 1.2.6), as the primary amine of Tris quenches the remaining amino reactive groups.
Following the protocol stepwise leads to a homogenous distribution of Fn on the silanized glass surface (see Figure 2), leaving a dimeric form of the protein. This resembles Fn´s structure in solution and is consistent with previous AFM data on other sample surfaces37. In addition, an appropriate concentration of RrgA on the AFM tip is obtained, which generates a target value of ~20% of well-defined interaction events during SMFS measurements (Figure 3 and Figure 4). Another elegant way to control the amount of molecules coupled to the sample substrate and cantilever tip besides varying the protein concentration and/or incubation times, is the combination of silane-agents with different secondary functional groups. By changing the ratio of protein reactive groups extending from the PEG-polymer, the number of immobilized proteins can be controlled15,16,17,18.
The protocol described here can also be used to immobilize other -NH2 containing molecules or be adjusted to couple proteins to other silicon-oxide surface besides glass and silicon nitride. Depending on the protein design, the amine reactive carboxyl group can be changed to a sulfhydryl reactive group (e.g., maleimide or ortho-pyridyl disulfide) to couple the protein via its free –SH groups. For Fn, this results in a predefined orientation13,17,20.
In summary, this protocol can be adjusted to serve different requirements and is suitable for other biophysical applications besides single molecule force spectroscopy experiments.
The authors have nothing to disclose.
TB and HG acknowledge financial support through the European Research Council "Cellufuel, Advanced Grant No. 294438". HCS acknowledges financial support from the Federal Ministry for Education and Research through the Innovationsallianz Technofunktionale Proteine (TeFuProt), SS acknowledges financial support from the Bavarian State Ministry for Science and Education through the research focus "Herstellung und biophysikalische Charakterisierung dreidimensionaler Gewebe – CANTER". We thank Conny Hasselberg-Christoph and Martina Hörig for technical support
Material | |||
2-Propanol | Carl Roth | 6752 | |
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide | Sigma-Aldrich | 03450 | EDC |
Acetic acid | Carl Roth | 3738 | 100 %; analytical purity |
Doubly distilled water | |||
Ethanol | Carl Roth | 9065 | ≥ 99.8 %; analytical purity |
Ethoxy silane polyethylene glycol acid | Nanocs | PG2-CASL-5k | 5 kDa; COOH-PEG-Si(OC2H5)3 |
Hydrochloric acid | Carl Roth | X896 | 32 % |
N-Hydroxysuccinimid | Merck | 804518 | NHS; for synthesis |
Phosphate Buffered Saline – Dulbecco | Biochrom | L1825 | PBS |
Probe molecule e.g. Fibronectin, human plasma | Sigma-Aldrich | F1056 | |
Probe molecule e.g. RrgA | Produced in laboratory | ||
Sodiumchlorid | Carl Roth | 9265 | NaCl |
Tris(hydroxymethyl)-aminomethan | Carl Roth | AE15 | ≥ 99,3 %; TRIS; Buffer Grade |
Name | Company | Catalog Number | Comments |
Equipment | |||
Beakers | |||
Glass cutter | |||
Glass slides | Carl Roth | 0656 | |
Inert gas desiccator | Sicco | ||
Inverted Microscope – Zeiss Axiovert 200 | Zeiss | ||
JPK NanoWizard 1 | JPK Instruments | ||
JPK NanoWizard SPM and DP software | JPK Instruments | ||
Laboratory oven | Binder | ||
Magnetic stirrer | IKA | ||
Micro spatula | |||
Microcentrifuge tubes | |||
Microsoft Excel | Microsoft | ||
Parafilm M | Brand | 701606 | |
Petri dishes | |||
pH-meter | Knick | ||
Pipettes | Starlab | 10-100 µl, 50-200 µl, 100-1000 µl | |
Precision balance | Acculab | ||
Silicon nitride cantilever – MLCT | Bruker AXS S.A.S | Spring constant ≤ 100 pN/nm | |
Sonication bath | Bandelin | ||
Staining jar | |||
Stereo microscope – Zeiss Stemi | Zeiss | ||
Stir bar | |||
Kimtech science precision wipes | Kimberly-Clark | ||
Twezzers | |||
UV PenRay | UVP, LLC | 90-0012-01 | Mercury spectrum with the primary energy at 254 nm |
Vacuum desiccator | |||
Vacuum pump | |||
Vortex mixer | VWR | ||
Weighing paper | Carl Roth | TP64 |