Abstract

Platform design

A sketch of our platform is depicted in Figure 1a: We use a bottom-up approach based on the self-assembling properties of streptavidin (SA) molecules to form regular two dimensional arrays on mica-supported fluid lipid bilayers containing biotinylated lipids^26–28. These arrays then serve as porous support for the upper reconstituted lipid bilayer. It is generated by spreading proteoliposomes that contain a small fraction of biotinylated lipids (5 %). First, the homo-tetrameric SA self-assembles on a lower supported fluid biotinylated planar lipid bilayer into highly ordered P2 symmetric crystalline arrangements (Fig. 1b) comprising a unit cell characterized by a=b=5.90 ± 0.07 nm and γ=90 ± 1° (Fig. 1c). Two of its monomeric biotin-binding subunits are thereby oriented towards the bilayer and occupied by biotin that is tagged to the dioleoyl phosphatidyl ethanolamine (DOPE) lipids, while the other two subunits are unoccupied facing away from the support. These subunits are accessible to high resolution HS-AFM imaging (Fig. 1c), and can be further used as specific sites for binding of biotinylated entities, as demonstrated for various soluble proteins²⁷.

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Figure 1

Platform for transmembrane protein observation.

a, Sketch of the Streptavidin-crystal periodically suspended membranes (SA-crystal PSMs) containing transmembrane proteins (e.g. SecYEG). b, A SA-crystal was formed on a lipid bilayer containing biotinylated lipids (DOPC/DOPS/biotin cap DOPE 6:2:2 w/w) as described¹⁶. c, High resolution imaging of the SA-crystal reveals two-fold symmetry and a unit cell a=b=5.90 ± 0.07 nm; γ=90 ± 1°. d, SecYEG containing proteoliposomes were incubated for ~ 3 min. forming lipid patches on top of the SA-crystal. Moderately diffusing SecYEGs could be observed.

The SA-crystal also provides small buffer-filled compartments that occupy ~ 37 % (volume~ 4.5 x 2.6 x 4 nm³, estimated from Fig. 1c and the height of the SA-crystal) of the unit cell area. Proteoliposomes containing either SecYEG or GlpF (for purification and reconstitution details cf. methods section and Supplementary Fig. 1) spread on the SA-crystal and fused to large periodically suspended membranes (PSM). Spreading was due to the specific interactions between SA and 5% of biotinylated DOPE lipids that were also included into the proteoliposomes. The size of the PSM patches is sensitive to the concentration of the proteoliposome suspension and the timespan it is applied to the SA-crystal (cf. Supplementary Fig. 2). The resulting membranes appear flat and stable, with a thickness of ~ 4 nm, indicating that they closely adhere to the underlying SA-crystal. The presence of SecYEG and GlpF could be recognized in HS-AFM movies (Supplementary Movie S1) as characteristic, spike-like topographical noise indicative for fast diffusing objects¹⁶. These fast lateral dynamics suggest that interactions with the support are vastly reduced. To fully resolve membrane proteins by HS-AFM, it is necessary to reduce their mobility to match the instrument’s imaging capabilities that allows us to capture diffusional processes of up to a few hundreds of nm²s^{-1 6–8}. Therefore, we introduced additional membrane obstacles in the form of glutaraldehyde (GA) crosslinks of neighboring SA molecules subsequent to SA-crystal formation (cf. methods). After spreading proteoliposomes on the SA-crystal, we finally observed membrane proteins of different mobilities (in this case SecYEG) in this uppermost bilayer (note that the incubation time interval used for Fig. 1d was chosen to be small in order to demonstrate the layer-by-layer composition of the platform; Full coverage: Supplementary Movie S2; Supplementary Fig. 2).

Apparent protein abundancies on SA-crystals and mica SLBs

The extent of chemical fixation of SA arrays prior to the final membrane deposition process can be used to fine-tune the lateral mobility of embedded proteins, as examined for SecYEG^{19, 23} and GlpF^{24, 25}. PSMs were generated on SA-crystals that were treated for a defined time (5 min) with different GA concentrations. Apparent protein abundance per scan area was then determined by HS-AFM imaging (Supplementary Movie S2, cf. methods) and compared to the corresponding values from conventional mica SLBs (Fig. 2a). The number of SecYEG detected via HS-AFM depended on the GA exposure during SA-crystal preparation. At the highest GA concentration, it twofold exceeded the number observed in SLBs on mica (black dashed line). In contrast, the abundance of GlpF detected by HS-AFM did not depend on glutaraldehyde treatment of SA-crystals, but resembled the value obtained from mica-SLBs (orange dashed line). For both SecYEG and GlpF, only proteoliposomes from the same reconstitutions were compared and therefore all membranes should have contained the same amount of proteins. The discrepancy in the apparent SecYEG abundancies can be rationalized by assuming that (i) ~50% of the SecYEG molecules on mica SLBs diffuse too fast to be captured by HS-AFM, and (ii) the SecYEG molecules on SA-crystals slow down thus enabling their identification and counting by HS-AFM imaging (which was not possible for PSMs on SA-crystals without GA treatment, compare Supplementary Movie S1). As the SecYEG and GlpF molecules we have used throughout this study carried a fluorescence dye (ATTO 488, cf. methods) we could test this hypothesis, by characterizing the mobility of SecYEG and GlpF that were reconstituted into mica SLBs or SA-crystal PSMs (10 mM GA) by means of fluorescence single particle tracking. Single particle trajectories were recorded and examined (cf. methods). On mica SLBs the data was best fit by a model²⁹ including two independent mobility components. Following the suggestion of Occam’s razor, we abstained from using models with a larger number of components, although they would also be compatible with the data. The analysis of trajectories (Supplementary Fig. 3) revealed that ~44% of SecYEG exhibited a high mobility of D=9.8 ± 3.3 x 10⁴ nm²s^-1 that is indeed too fast to be captured by HS-AFM (limited up to a few hundreds of nm²s^{-1 6–8}), whereas the remaining fraction (~56%) had a lower mobility of only D=0.2 ± 0.1 x 10⁴ nm²s^-1. Fluorescence measurements of SecYEG in SA-crystal PSMs, GlpF in mica SLBs, and GlpF in SA-crystal PSMs only detected a single slow fraction. The excessive GA concentration for SA fixation was responsible for protein slow-down in SA-crystal PSMs (compare next paragraph).

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Figure 2

Membrane protein mobility depends on extent of SA-crystal chemical fixation.

a, Apparent membrane protein abundance per scan-area (A_S=400 x 400nm²) in SA-crystal PSMs (left panel) vs. mica SLBs (right panel). b, Fraction of detected proteins exhibiting an intermediate mobility. c, Lateral diffusion constants of intermediate and slow fractions as a function of glutaraldehyde exposure during SA-crystal preparation. All values are mean ± s.e.m.

Tailoring membrane protein mobility

HS-AFM imaging of SecYEG and GlpF revealed two fractions: An intermediate and a slow mobility fraction. Increase of GA concentration during the SA-crystal fixation step decreased the intermediate mobility fraction and increased the slow mobility fraction, respectively. To characterize both lateral mobility and the respective fractions, we performed a quantitative analysis of the corresponding HS-AFM movies by generating SecYEG and GlpF trajectories and subjected the ensemble to a diffusional analysis again using a model²⁹ including two independent mobility components (cf. methods; Supplementary Fig. 4): the first population of molecules possessed an intermediate mobility (i.e. they were less mobile than the fast molecules that were observed only by fluorescence), while the mobility of the second population was so low that the calculated diffusion constant was smaller than the positional accuracy. Nevertheless, we do not call them immobile because we observed molecules switching from intermediate to low mobility and back (Supplementary Movies S3 and S4). Depending on GA concentration during SA-crystal preparation, the average fraction of intermediately mobile proteins was tuned from ~25 - 30% (0.4 mM GA) to ~0 – 8% (10 mM GA), Figure 2b. Similarly, the mobility of these fractions was reduced from 80 nm²s^-1 to ~ 1 nm²s^-1 (SecYEG) and 40 nm²s^-1 to 10 nm²s^-1 (GlpF) when SA-crystals were treated with increasing GA concentrations (Fig. 2c). Fluorescence recovery after photo bleaching (FRAP) and single particle tracking experiments (cf., methods; Supplementary Fig. 5) served to assess membrane quality and fluidity of SA-crystal PSMs. The fluorescent lipids recovered homogenously with a diffusion coefficient of ~ 1.5 μm²s^-1 (determined from FRAP and single particle trajectories, Supplementary Fig. 5) indicating the presence of continuous SA-crystal PSMs.

Crowding is responsible for confinement into nanodomains

Without GA treatment of the SA-crystal, membrane proteins moved too fast to be fully resolved by HS-AFM (Supplementary Movie S1). A minimal treatment of 0.4 mM GA was necessary to effectively slow down their diffusive motion. Furthermore, a close inspection of trajectories (Fig. 3a and b, Supplementary Movie S3 and S4) revealed that the proteins did not randomly diffuse but that they were confined into nanodomains: The proteins repeatedly moved bi-directionally along the same tracks. The lateral dimension of these domains depended on the GA concentrations, as indicated by the loss of the intermediately mobile protein fractions at the highest GA concentrations (Fig. 2b and c). A high resolution image of a SA-crystal (Fig. 3c; white and yellow axes) reveals the mechanism for the observed dynamics: The contact region between two adjacent SA molecules is not planar but features a groove that connects the holes in the SA-crystal, thereby allowing flexible membrane-protruding moieties¹³ to pass. As this groove propagates throughout the SA-crystal (white and yellow axes, Fig.3c), we suggest that GA treatment effectively introduces obstacles within these grooves by covalently interlinking amine groups. We have generated a model of a SA-crystal (Fig. 3d; based on the SA structure pdb 3RY2³⁰, and lattice constants, Fig. 1c) and identified lysine and arginine residues within flexible loops on the SA surface as potential cross-linking sites within the groove at the interface between neighboring SA molecules (Fig. 3e and f). Without crosslinking, the parts of SecYEG and GlpF protruding the membrane are likely to slip between adjacent SA molecules, thereby retaining high lateral mobility. The passage of membrane protruding parts of SecYEG and GlpF became increasingly restricted as the number of crosslinks increased. Starting from a certain GA threshold concentration the tracks are no longer connected to other ones. That is, proteins are trapped within these GA-induced nanodomains and within these, their motion is restricted by the discrete template provided by the meshwork of holes and obstacle-free grooves between adjacent SA molecules. This results in trajectories that have parallel and perpendicular components (in accordance with the underlying grooves) as shown in Figure 3a and b, as well as in Supplementary Movie S3 and S4. As GA concentration is further increased the lateral dimension of nanodomains further shrinks so that finally all membrane proteins belong to the slow mobility fraction (Fig. 2).

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Figure 3

Confined lateral mobility of membrane proteins.

a, Overlay of trajectories of SecYEG embedded in a SA-crystal PSM, Supplementary Movie S3. The proteins repeatedly diffuse along the same pathways. b, Overlay of trajectories of GlpF embedded in a SA-crystal PSM, Supplementary Movie S4. Similar to SecYEG, the proteins repeatedly diffuse along the same pathways. c, 3D representation of the high resolution image of a SA-crystal (Fig. 1c). The meshwork of grooves between adjacent SA molecules is indicated by the yellow and white axis. d, Molecular model of the SA-crystal. e, Magnification of the SA-SA interface (top view). Potential sites (amine groups) for Glutaraldehyde crosslinking of neighboring SA molecules are displayed (red circles). f, Side view on the SA-SA interface. Amino acids relevant for potential glutaraldehyde cross-links are indicated. Colorcode indicates their location on SA1 (blue), SA2 (yellow), and SA3 (green).

Topographical and functional assessment of membrane proteins

The GlpF and SecYEG proteins were examined for their functionality in standard in vitro assays. GlpF was proven to be active by measuring its water permeability from osmotically challenged vesicles in a stopped flow assay¹⁴, and SecYEG was subject to a standard translocation assay³¹. We further analyzed the size (heights) of the membrane-protruding proteinaceous parts with respect to the surrounding lipids to determine their orientation (periplasmic/cytoplasmic surface up/down) in mica SLBs and SA-crystal PSMs. A probability density function (pdf) of height values was generated from height measurements of membrane protruding GlpF moieties on mica SLBs (dashed line) and SA-crystal PSMs (solid line, Fig. 4a, left panel). A detailed analysis (Supplementary Fig. 6a and b) revealed that the pdfs were best fit by a sum of three Gaussians (I-III). Comparison to the crystal structure (Fig. 4a, right panel) shows that for the SA-crystal PSMs, the mean values of these Gaussians match the heights of membrane protruding ends of transmembrane helices at the cytoplasmic side (I; fraction 0.40 ± 0.02), as well as the alpha-helical protrusions at the periplasmic side (II; fraction 0.44 ± 0.02). Occasionally (fraction 0.16 ± 0.03), some molecules appeared higher (III), very likely reflecting the unstructured, very flexible 40aa linker masking the lower protrusions (I) at the cytoplasmic side. In total, 56 % of molecules were oriented with the cytoplasmic side (I+III) and 44 % with the periplasmic side (II) facing upwards. For mica SLBs (Fig. 4a, dashed line), the shape of the pdf was comparable, however, the relative orientation of molecules was slightly different, with 43% of molecules oriented with the cytoplasmic side (I+III) and 57 % of molecules with the periplasmic (II) side facing upwards. The quality of high resolution images obtained from GlpF tetramers embedded in SA-crystal PSMs (immobile, Fig. 4b, and mobile, Supplementary Movie S5) was comparable to that of images of GlpF embedded in mica SLBS taken previously¹³. A similar height-analysis was done for SecYEG (Fig. 4c, left panel; Supplementary Fig. 6c and d). For SA-crystal PSMs, the pdf (solid line) was best fit by a sum of three Gaussians. Comparison to the crystal structure (right panel) revealed that the mean values of Gaussians match the heights of secondary structural elements protruding from the membrane at the periplasmic side (I; fraction 0.47 ± 0.06), as well as the lower protrusion (II, fraction 0.43 ± 0.05) and the less frequently detected (since more flexible), higher protrusion (III, fraction 0.10 ± 0.04) both at the cytoplasmic side. In total, 47 % of proteins detected in SA-crystal PSMs were oriented with the periplasmic side upwards (I) and 53 % of molecules (II+III) were oriented with the cytoplasmic side facing upwards. For the mica SLBs, the pdf (dashed line; Fig.4c, left panel) was best fitted by a sum of two Gaussians (Supplementary Fig. 6d). The mean value of the first Gaussian (I; fraction 0.73 ± 0.03) again matches the height of the periplasmic protrusion and the mean value of the second Gaussian (II) was found to be in between the values identified for the cytoplasmic protrusions of SecYEG in the SA-crystal PSMs and occurred less frequent (0.27 ± 0.03) than these. In total 73 % of the proteins detected in mica SLBs were oriented with the periplasmic side upwards and 27 % of molecules were oriented with the cytoplasmic side facing upwards. As the overall number of proteins detected in mica SLBs was ~ half the number detected in SA-crystal PSMs (cf. Fig. 2a) this 27% represent only 13.5% of the overall protein content suggesting that the majority of proteins with the cytoplasmic side facing upwards (37.5% of total protein content) are included in the fast-diffusing fraction as determined by single particle tracking (Supplementary Fig. 3), and are thus too fast for HS-AFM observation. As these protrusions are representing the binding site for the translocase motor protein SecA^{32, 33} (post-translational translocation) and the ribosome¹⁹ (co-translational translocation), SecA binding to this fraction cannot be observed by HS-AFM either.

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Figure 4

Characterization of membrane proteins embedded in SA-crystal supported lipid bilayers.

a, Probability density function (pdf) of heights of membrane protruding GlpF moieties measured on mica (n=206) and SA-crystal (n=389; 10 mM glutaraldehyde;) (left panel). The pdfs were best fit by a sum of three Gaussians (cf. Supplementary Fig. 6a and b). The mean values of these Gaussians (I-III) match the heights of structural features relative to the membrane as known from crystal structure (right panel, pdb: 1fx8²⁴ + unstructured 40 aa tag on the cytoplasmic side^{13, 14}, cf. methods). b, HS-AFM time series of a single GlpF tetramer (periplasmic side)¹³ embedded in a SA-crystal PSM. c, Probability density function (pdf) of heights of membrane protruding SecYEG moieties measured on mica (n=697) and SA-crystal (n=1232; 10 mM glutaraldehyde;) (left panel). The pdf for mica SLBs was normalized so that the area under the curve reflects the fraction of proteins observed on mica relative to the number of proteins observed on SA-crystals per scan area (Fig.2a). The pdfs were best fit by a sum of three (SA-crystal) resp. two (mica) Gaussians (cf. Supplementary Fig. 6c and d). The E. coli SecYEG structure (right panel, pdb: 3J45¹⁹) suggests three different height populations (I at the periplasmic side, II and III at the cytoplasmic side) that can be collectively found in the height pdf collected from SecYEG embedded in the SA-crystal PSM (cf. Supplementary Fig. 6c). For the mica SLBs, the pdf (dashed line; Fig.4c, left panel) was best fitted by a sum of two Gaussians (Supplementary Fig. 6d) whereas the cytoplasmic fractions (II+III) were far less pronounced (arrow, cf. Supplementary Fig. 6d). A height pdf of SecA (n=134) bound to SecYEG reveals a pronounced height increase of up to 2 nm compared to the bare SecYEG (cytoplasmic side). Topographical noise introduced by the lipid associated, fast diffusing SecA prevented the detection of the lower height populations. d, HS-AFM time series taken from Supplementary Movie S6 of SecA (white circles) bound to SecYEG. Only very rarely could the dissociation of SecA from SecYEG be observed uncovering the underlying SecYEG channel.

Catching the act of ligand binding to membrane receptors

However, on SA-crystal PSMs, this fraction was accessible to HS-AFM imaging, so that SecA being bound to SecYEG was observed (Fig. 4d). SecYEG-containing SA-crystal PSMs were first incubated with 2 μM SecA for 2 min, then rinsed to remove unbound SecA and subsequently subjected to HS-AFM imaging (Fig. 4d). The SecA particles protruded up to 4 nm from the SA-crystal PSMs (Fig. 4c, red curve) and were stably bound to SecYEG (Fig. 4d) which is in agreement with surface plasmon resonance measurements of SecA binding to SecYEG in inner membrane vesicles³⁴. This confirms that the involved epitopes on SecYEG³³ are structurally intact and functional, and that the involved rearrangements of SecYEG transmembrane helices necessary to facilitate this association³³ can occur without hindrance. On rare occasions SecA was released, thereby exposing the SecYEG molecule that was underneath (Fig. 4d, circle). Sometimes we observed its subsequent re-association (Supplementary Movie S6). In addition, we found relatively high topographical ‘spike’ noise¹⁶ under these conditions that we attributed to lipid bound SecA³⁵. These SecA molecules migrated along the membrane surface too fast (apparent lateral diffusion coefficient D=1.86 ± 0.16 x 10⁶ nm²s^-1; assessed by FRAP) to be fully resolved by HS-AFM imaging.

Conclusions

Protein reconstitution into SA-crystal PSMs allowed us to mimic the crowded environment of biological membranes. In this way, we have tuned the mobility of membrane proteins with protrusion sizes ranging between ~ 0.7 – 3 nm (13 to 51 residues) which covers the vast majority of extra-membraneous domains of integral membrane proteins found throughout eubacterial, archaean, and eukaryotic organisms³⁶. While retaining their natural lateral and rotational mobility, reconstituted membrane proteins can now be imaged at sub-molecular resolution by virtue of their transient confinement into nanodomains that are similar in size to rafts or other cytoskeleton assisted nanodomains.

Thus SA-crystal PSMs offer the possibility to use high speed atomic force microscopy³ to capture (i) dynamic conformational changes of single membrane and soluble proteins^{37, 38} alike and (ii) interactions of soluble proteins and membrane proteins with nm-spatial and millisecond temporal resolution.

The technique may also be of interest to conventional AFM as the proteins may be rendered practically immobile without being tethered or being in contact to the support. The platform is well suited for studying reconstituted membrane proteins not only by AFM but also by other techniques - like by optical microscopy (e.g. single dye tracing and fluorescence resonance energy transfer), surface plasmon resonance, or quartz crystal micro balance. It thus eases the investigation of interactions between membrane proteins and peripheral proteins, or compartmentalized biochemical processes.

Confinement of mobile membrane proteins into small patches is also possible by suspending lipid bilayers on highly ordered functionalized pore arrays in silicon nitride. The main limitation here is not the pore size that can be reduced below the commonly used ~ 1 μm³⁹, but is rather represented by the strict border of these domains that no membrane protein can pass. In contrast, the nanodomains in SA-crystal PSMs can be tuned so that the confinement is transient, i.e. that a certain probability of protein escape remains. In this sense, they are a much better model of membrane rafts, which allow for protein and lipid exchange with their environment. It is also imaginable that the incorporation of photo-reactive amino acids into SA molecules at locations that form the SA-SA interfaces within the SA crystals might offer the possibility to use a patterned photo-crosslinking approach to generate domains with specifically tailored sizes, orientations, and shapes.

The combination of SA-crystal PSMs with HS-AFM will further deepen our knowledge about the dynamic structure of membrane proteins and their interplay with peripheral proteins. It will enable us investigation of receptor activation by peripheral ligands that migrate along the membrane surface to find their target. In the particular case of bacterial translocation machinery, it puts the direct visualization of protein translocation within tangible reach.

Methods

Supplementary Material

Acknowledgments

Footnotes

References