Letter pubs.acs.org/NanoLett
Localizing Chemical Groups while Imaging Single Native Proteins by High-Resolution Atomic Force Microscopy Moritz Pfreundschuh,‡,⊥ David Alsteens,‡,⊥ Manuel Hilbert,§ Michel O. Steinmetz,§ and Daniel J. Müller*,‡ ‡
Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villingen, Switzerland
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S Supporting Information *
ABSTRACT: Simultaneous high-resolution imaging and localization of chemical interaction sites on single native proteins is a pertinent biophysical, biochemical, and nanotechnological challenge. Such structural mapping and characterization of binding sites is of importance in understanding how proteins interact with their environment and in manipulating such interactions in a plethora of biotechnological applications. Thus far, this challenge remains to be tackled. Here, we introduce force−distance curve-based atomic force microscopy (FD-based AFM) for the high-resolution imaging of SAS-6, a protein that self-assembles into cartwheellike structures. Using functionalized AFM tips bearing Ni2+-N-nitrilotriacetate groups, we locate specific interaction sites on SAS-6 at nanometer resolution and quantify the binding strength of the Ni2+-NTA groups to histidine residues. The FD-based AFM approach can readily be applied to image any other native protein and to locate and structurally map histidine residues. Moreover, the surface chemistry used to functionalize the AFM tip can be modified to map other chemical interaction sites. KEYWORDS: Biomolecular bonds, chemical recognition imaging, multiparametric imaging, force spectroscopy, ligand−receptor interaction, single-molecule imaging
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Interestingly, FD-based AFM can also detect specific interactions that are exposed on biological samples.8,9,11,14,25−28 To measure these interactions, FD-based AFM records the interaction forces between a chemically functionalized AFM tip and a sample (Figure 1a−c). This approach allows the direct correlation of a sample topography with structurally localized mechanical properties and a specific interaction map.25 Recent examples have used AFM tips functionalized with specific bioligands to image live cells and at the same time to locate cell surface receptors,29,30 functionalized with chemical groups to map the chemical properties of cell surfaces,31 and functionalized with specific lectin to map N-acteylgalactosamineterminated glycolipids on red blood cells.28 In these examples, FD-based AFM was also used to quantify the force required to rupture particular biochemical interactions. Remarkably, analysis of the force required to separate biochemical bonds (e.g., receptor−ligand bonds) can provide insight into their kinetic, energetic, and mechanical properties.32,33 However, although powerful FD-based methods using functionalized AFM tips to locate and characterize specific interactions have been impaired
riginally conceived as an imaging tool, atomic force microscopy (AFM)1 rapidly developed into a multifunctional toolbox enabling researchers to observe biological systems including tissues, cells, membranes, and single molecules, and to manipulate these systems with unprecedented precision.2−6 More recently, the AFM has matured into a multiparametric tool that allows the imaging of biological systems while simultaneously mapping their mechanical properties to subnanometer resolution.7−11 The imaging and quantification of mechanical properties of biological samples has been much simplified by force−distance (FD) curve-based AFM (FD-based AFM), which records FD curves for every pixel of the AFM topograph (Figure 1a).11 For each FD curve, the AFM tip is approached to contact and is withdrawn from the biological sample. The deflection of the AFM cantilever detects the interaction forces between tip and sample (Figure 1b).12 These interaction forces enable the quantification of adhesion, deformation, Young’s Modulus, energy dissipation, and other parameters that mechanically characterize the biological sample. Because FD-based AFM records one or more FD curves for every pixel of the sample topograph, these properties can be directly correlated to the structure of the biological system under investigation.10,11 Recently, FD-based AFM has been applied to measure mechanical properties of cells,7−9,13,14 viruses,15 protein membranes,16,17 lipid membranes,18,19 proteins,16,17,20,21 and fibrils.22−24 © 2014 American Chemical Society
Received: April 8, 2014 Revised: April 22, 2014 Published: April 28, 2014 2957
dx.doi.org/10.1021/nl5012905 | Nano Lett. 2014, 14, 2957−2964
Nano Letters
Letter
Figure 1. Locating and quantifying specific interactions using FD-based AFM. (a) Pixel-for-pixel FD-based AFM approaches and retracts the tip of an AFM cantilever from the sample to record interaction forces, F, over the tip−sample distance in FD curves. The high precision of the approach allows detection of forces with piconewton sensitivity and pixel sizes 100 pN. (c) Mapping of all adhesion peaks to the topograph localizes the C-terminal ends of the SAS-6 coiled coil rod domains with improved accuracy. AFM images were taken in imaging buffer containing 1 mM NiSO4 using a Ni2+-NTAmodified AFM tip at ∼27 °C. AFM topographs show color (a) and gray (c) scales corresponding to a vertical range of 6 nm. Dashed circles highlight topographic regions at which specific adhesion events were detected.
second control experiment, the AFM tip was functionalized with only PEG groups (Figure 3g). Again, the high-resolution AFM topograph showed individual SAS-6 cartwheels with their emanating coiled coil rods, and the adhesion map detected low adhesion forces (4 nm indicative of the stretching of the PEG linker and of the coiled coil rods bearing the His6-tags. Additionally, these adhesive events were not associated with SAS-6 cartwheels. This result confirms that the stronger adhesive interaction forces shown in Figure 3a−c are specific and originate from interactions between the Ni2+-NTA groups attached to the AFM tip and the His6-tags located at the C-terminal end of the SAS-6 coiled coil rod domains. Improving the Adhesion Maps of His6-Tags Fused to SAS-6 Molecules. After having demonstrated that AFM tip functionalized with Ni2+-NTA groups can be employed to detect His6tags in SAS-6, we sought to improve the recording of adhesion maps. One disadvantage of these maps is that the number of specific adhesion events detected is rather low (see Discussion above). The simplest way to increase the number of adhesion events and thereby to improve the detection of histidine residues is to increase the number of FD curves recorded. In addition, we also wanted to investigate whether the histidine residues of SAS-6 can be repeatedly detected. Therefore, we decided to record multiple high-resolution adhesion maps of SAS-6 cartwheels (Figure 4). The adhesion events from three consecutive adhesion maps show that in most cases (88.2% (31 of 34)) adhesion was repeatedly detected (at least twice) at the same structural regions (Figure 4b). Only a few distal ends of the coiled coil rods showed no adhesive interaction. The mapping of these adhesion events to the high-resolution topograph reveals their localization to the C-terminal ends of the SAS-6 coiled coil rods (Figure 4c). This experiment demonstrates that interactions can be detected reproducibly. It further indicates that some histidine side chains of SAS-6 are not accessible to bind to the functionalized AFM tip whereas a few histidine side chains were accessible only occasionally. We know
These adhesion events, which were clearly unlike those measured on the mica support, were judged to be specific. Rarely, we detected comparable adhesion forces in the contact region between tip and sample (
