Controlled Mechanical Actuation of Adsorbed Forisome Mechanoproteins: A Step Toward Biomolecular Devices

Forisomes are giant structural plant mechanoproteins that reversibly undergo a transition from a longitudinally expanded to a contracted (dispersed) state. This transition is driven by a change in the concentration of Ca2+ ions. Artificial forisomes, expressed in yeast, have a width of 0.7 µm and a length of 4.6 µm and adsorb onto a gold surface. Scanning electrochemical microscopy imaging of the adsorbed forisomes reveales a heterogenous film in which several proteins adsorb together on the substrate surface. The adsorbed forisomes maintain their biological activity and undergo reversible contraction–expansion reactions. Infrared spectroscopy (IRS) shows that Ca2+ ions are coordinated to the carboxylate groups in the side chains of Asp and Glu affecting the 3D arrangement of the corresponding protein fragments allowing for a longitudinal contraction, dispersion of the forisome volume, and water uptake. In films, expanded forisomes accumulate small ions (ethylendiaminetetraacetic acid dianion or [Fe(CN)6]4‐) that are released upon Ca2+ driven dispersion. The properties described here make forisomes attractive for biotechnological applications.


Introduction
[3] The DOI: 10.1002/adfm.202312159term "forisome" is derived from the Latin word foris, meaning gate wing, and the Greek word soma, meaning body.The nomenclature refers to the function of these proteins, as they block the transport of sugars and other vital substances such as amino acids, ribonucleic acids and signaling substances in the phloem in response to injury and attack by pests that feed on the phloem. [2]In this process, forisomes block the fluid transport by transforming their normally tightly packed, spindleshaped structure (longitudinally expanded state) into a dispersed plug (longitudinally contracted state), thus preventing the loss of energy-rich plant sap on the one hand and the penetration of plant pathogens on the other.An anisotropic conformational change of the forisomes is characterized by a longitudinal contraction of ≈30%, a radial swelling of up to 180% and an increase in volume of up to nine times due to water influx into the forisome. [2,4]The mechanical activation of forisomes is remarkably independent of the plant's energy source, adenosine triphosphate (ATP), and is solely triggered by an increase in the concentration of Ca 2+ ions in the cells. [1,2]The Ca 2+ ions enter the cell either through injury or as part of a signaling cascade. [2,5]When the cells of the plant's vascular system are able to regenerate, the forisomes return to their original spindle-shaped expanded state and allow mass flow to resume.The biological activity of the protein is therefore described by a reversible mechanical response: the contraction and expansion.The plant cell provides a reducing environment for the forisomes and oxygen has been shown to inhibit the conformational change of the protein complexes. [4]However, this inhibition can be prevented in vitro by the addition of a chemical O 2 -scavenging system of NaHSO 3 as reductant and Co 2+ as catalyst.
Forisomes can measure between 10 to 55 μm in length and 1 to 5 μm in width, rendering them visible even under a light microscope. [3,6]They consist of several million different subunits that assemble into a supramolecular protein complex.In the model plant Medicago truncatula, these subunits are known as MtSEO-F1, F2, F3 and F4, which stand for Sieve Element Occlusion by Forisomes. [7]This nomenclature reflects the function of the assembled protein complex.[11][12] The smallest identified and best characterized building block of forisomes is MtSEO-F1, a protein with a mass of ≈75 kDa that is composed of three domains: the N-terminal domain, a potential thioredoxin fold and a C-terminal domain.MtSEO-F1 monomers aggregate to dimers.Through hydrophobic interactions of their N-terminal protein domains, the dimers expand to form a smallest substructure of the forisomes, the filaments.Two of these filaments, stabilized by non-covalent interactions, wind helically around each other to form fibrils. Several fibrils assemble into fibril bundles, whose cohesion is stabilized by cysteine bridges.The quaternary structure of the protein protects the disulfide bridges from the reducing environment of the cell.The fibril bundles assemble in a similar manner to create fibers, several of which contribute to the formation of the protein bodies.
Purification of large quantities of forisomes from plant tissue is a labor-intensive and time-consuming process, which is a major drawback for elucidating their structure and reactivity.A breakthrough was the production of substantial quantities of forisomes within yeast cells. [13,14]In this context, only one MtSEO-F subunit (either MtSEO-F1 or F4) is necessary for the assembly of so-called artificial forisomes from millions of these subunits.In addition, the size of the MtSEO-F1-based artificial forisomes produced in yeast is in the range of 4.6 μm × 0.7 μm.This makes them smaller than the forisomes found in plants. [14]he ability to produce soluble MtSEO-F1 subunits in bacterial cells [10] and artificial forisomes in yeast facilitated their structural analysis.Infrared spectroscopy is an attractive analytical method to elucidate in situ the secondary structure and its changes in proteins embedded in various environments. [15,16]Spectroscopic studies shed light on the secondary structure of the MtSEO-F1 subunits showing that helices constitute the major secondary structure element of forisomes. [11]Furthermore, these studies revealed that soluble MtSEO-F1-dimers in aqueous solution do not show any structural changes in the presence of Ca 2+ .Similarly, the molecular analyses of the MtSEO-F1 subunits did not reveal a distinct calcium-binding motif [17] suggesting that the 3D arrangement of the MtSEO-F1 subunits is an important factor for the response of forisomes to Ca 2+ .As elucidated by polarization modulation infrared reflection-absorption spectroscopy (PM IR-RAS), the adsorption of MtSEO-F1 subunits onto a gold surface results in higher substructures that are capable of undergoing conformational changes in the presence of Ca 2+ . [11]iven the straightforward production and cost-effective purification of artificial forisomes, along with their inherent structural stability and simple reaction mechanism, forisomes are exceptionally well-suited for applications in biotechnology.Interestingly, in vitro forisomes respond not only to a physiological trigger (Ca 2+ ) but also to other divalent cations, such as Ba 2+ and Sr 2+ , by a transition from an expanded (spindle) to a contracted and dispersed (plug) shape. [2]The addition of chelating agents like EDTA (ethylenediaminetetraacetic acid) and EGTA (ethylene glycol-bis(-aminoethyl ether)-N,N,N′,N′tetraacetic acid) permits the forisomes to revert to their original expanded state.Additionally, altering the physiological pH from 7.3 to either moderately acidic (pH 4.6) or basic (pH 9.6-10.9)triggers a similar response of forisomes, which is also reversible upon returning to the initial pH. [2]The ability of forisomes to respond to non-physiological triggers increases the scope for potential biotechnological applications.For example, the reversible response of forisomes to changes in pH facilitated the control of forisomes through electrotitration. [2]Through electroti-tration, a single forisome can undergo several thousand contraction/expansion cycles, underscoring the stability and longevity of the protein complexes.
The conversion of chemical energy into mechanical work predestines forisomes as biomaterials.Their inherent role in reversibly closing a plant tube system qualifies them for use as valves in microfluidic systems.Beyond this application, further biotechnological advances in understanding the self-assembly and actuation processes of (modified) forisomes could open up numerous applications in materials science, medicine, and biotechnology. [12,13,18]For instance, the forisomes surface can already be utilized to immobilize enzymes, benefiting from enhanced reaction cascades through substrate channeling. [19]The strategic placement of these protein bodies using fused tags also allows their utilization as compact bioreactors within lab-on-chip systems. [9]Precise modifications of forisomes using molecular biology methods could further expand their range of target ligands and vary their binding capacities, thus broadening the potential applications of these biomaterials. [12]Nevertheless, comprehensive investigations into both the immobilization and reactivity of forisome films are still necessary, for which this report provides a foundation.

Results and Discussion
For this study, artificial forisomes, further called forisomes, based on MtSEO-F1 subunits were selected.Forisomes were adsorbed onto gold surfaces from KClO 4 and NaCl electrolyte solutions.The type of electrolyte had a major impact on the amount and structure of the adsorbed forisomes as elucidated by means of PM IRRAS. Figure 1 shows PM IRRA spectra in the wavenumber (%) range of the amide I' vibration for a forisome film on a gold surface adsorbed from KClO 4 (Figure 1a) and NaCl (Figure 1b) electrolyte solutions.The spectra were recorded at the gold|air interface after removal of the sample from the adsorption solutions.The amide I vibration band appears in proteins between 1700 and 1600 cm −1 and originates predominantly from the (C═O) stretching mode (≈76%) with contributions from the (CN) stretching mode and in-plane bending of the NH groups. [15,20]The deconvolution of the amide I vibration (Supporting Information, Sections SI-S1 and SI-S2, Supporting Information) mode allows for the assignment of the secondary structure elements in proteins, [15,20] for which characteristic frequencies are listed in Table S1 (Supporting Information).To avoid spectral overlap of the amide I vibration with the OH deformation mode in H 2 O (1650-1600 cm −1 ), the electrolyte solutions were prepared with D 2 O.The assignments for amide I' and amide II' correspond to the amide vibration modes measured in D 2 O.
The amide I' band in forisomes adsorbed from KClO 4 electrolyte solution is weak and broad despite a 20 h long adsorption time.The second derivative of the IR spectrum in Figure 1a has several minima revealing that many overlapping modes contribute to the overall amide I' band.[22] The amide I' mode in Figure 1a is overlapped with an intense mode ≈1720 cm −1 that is associated with free, non-hydrogenbonded carbonyl groups.This signal arises from misfolded or even denaturated proteins. [15,22]Note, that the (C═O) stretching mode in protonated carboxylic acid side chains in Asp and Glu are expected above 1720 cm −1 and may also contribute to this absorption mode. [15] forisome film adsorbed from NaCl solution yields the IR spectrum in Figure 1b that significantly differs from that measured after adsorption from KClO 4 solution (Figure 1a).The amide I' mode is centered at 1648 cm −1 , indicating that -helices are the dominant secondary structure (c.f., Table S1, Supporting Information).This finding is in good agreement with the sequence predictions for forisomes provided in Section SI-S3 (Supporting Information), which suggests that forisomes adsorbed on the gold surface from NaCl solution retain their native secondary structure.The amide I' band in Figure 1b has a negligible intensity above 1700 cm −1 , which is probably caused by the (C═O) stretching mode in Asp and Glu side chains. [15]M IRRAS probes a large sample surface.Specifically, the spectra in Figure 1, were collected from an area of 1.5 mm × 12 mm.Therefore, the measured signals are a weighted average of different forisome structures that might exists side by side in this area.MtSEO-F1 forisomes expressed from yeast are ≈0.7 μm wide and 4.6 μm long. [14]This size exactly matches the spatial resolution of IR microscopy. [23]This technique was used to test the compositional and possibly structural heterogeneity of adsorbed forisomes.Heterogeneity is expected because the adsorbed protein layer may contain single forisomes as well as their larger agglomerates.The forisome film was ad-sorbed from 0.1 m KClO 4 solution, because this layer exhibits strong adsorption modes in the IR spectra that are well separated from the amide modes in forisome.Optical microscopy images (Figure S2, Supporting Information) show surface regions rich in protein (Figure S2a, Supporting Information) and regions of low protein content (Figure S2b, Supporting Information).The forisome films are heterogenous; many bodies assembly together in forisome-rich aggregates forming spots of a compact coating.
Figure S3 (Supporting Information) shows IR spectra with a probed area of 10 μm × 10 μm.They were obtained by attenuated total reflection infrared spectroscopy (ATR IRS) recorded with an IR microscope from two distinct areas of a forisome film on gold.The two spectra in Figure S3 (Supporting Information) differ significantly.The IR spectrum of the protein-rich surface region shows a strong amide I band with a maximum at 1642 cm -1 and amide II with a maximum at 1538 cm -1 (Figure S3a, Supporting Information).In contrast, the spectrum taken from a proteinpoor surface region shows only very weak and poorly resolved amide I and amide II absorption bands, but a strong absorption band from the perchlorate ions at 1150 cm -1 (Figure S3b, Supporting Information).These data show very clearly the heterogeneity in the composition of the adsorbed layer obtained from KClO 4 electrolytes.
Forisomes contract in vivo and in vitro after exposure to solutions containing Ca 2+ ions [2][3][4] and return to their expanded state upon decrease in the Ca 2+ concentration.The contractionexpansion reaction of forisomes is reversible and is defined as a reaction cycle.To study the associated changes in the secondary structure, the contraction of adsorbed forisomes was investigated in forisome films on Au surfaces adsorbed from NaCl electrolyte solution in D 2 O.As can be inferred from Figure 1b, the secondary structure of the MtSEO-F1 subunits constituting the forisomes is preserved in the resulting layers. [11]Noticeable spectral changes accompany the reaction cycle of adsorbed forisomes (Figure 2). Figure 2a shows the PM IRRA spectrum of the freshly adsorbed forisome film from NaCl electrolyte, Figure 2b was recorded after immersion of the film in 0.01 m CaCl 2 solution for 15 min, and Figure 2c was obtained after solution exchange and exposure to 0.1 m NaCl + 0.01 m Na 2 EDTA for 60 min.Na 2 EDTA was added to complex the Ca 2+ ions and to induce expansion of the forisomes.
In the absence of Ca 2+ , two strong and asymmetric IR absorption modes with maxima at 1650 cm −1 [amide I″+ as (COO − )] and 1420-1410 cm −1 [amide II″ +  s (COO − )] are present in the PM IRRA spectrum of expanded forisomes (Figure 2a,c).Deconvolution of the amide I' band revealed that -helices and -sheets contribute to the secondary structure elements of adsorbed forisomes.The IR absorption modes from the forisome film were the most intense in a freshly prepared film (Figure 2a).The intensities of all absorption bands decreased irreversibly after the first immersion of the protein film in a 0.01 m CaCl 2 solution, indicating the desorption of some forisomes from the gold surface in the contracted state.The integral intensity of the amide I' band in contracted forisomes differs from that of expanded forisomes (see Figure S4, Supporting Information).In the subsequent reaction cycles, the integral intensity changes reversibly between the two states indicating that the surface concentration of forisomes adsorbed on the gold surface is constant.The intensity of the absorption bands in PM IRRAS is sensitive to both the surface concentration of adsorbed species and their orientation in  an anisotropic film. [16]In this case, it is reasonable to assume a constant surface concentration because potentially desorbed forisomes would be discarded during a solution exchange and could not readsorb.At constant forisome surface concentration, a difference in the integral intensity of the amide I' band between the expanded and contracted forisomes points at a distinct average orientation of the helices and -sheets within the protein bodies.
Ca 2+ binding does not affect the elements of the secondary structure (see Figure 3).However, the wavenumber of the absorption maximum of the amide I' band of the -helices changes during the reaction cycle.In expanded forisomes (in the absence of Ca 2+ ), the -helices give the amide I' mode at 1649−1651 cm −1 (Figure 2a,c; Figure S5, Supporting Information).In contracted forisomes (in the presence of Ca 2+ ), the amide I' band of -helices is up-shifted to 1656 cm −1 (Figure S5, Supporting Information).In contracted forisomes an up-shift of the absorption maximum of the amide I' band of -helices indicates a general decrease in the hydrogen bond strength at the -helices.A transition from the expanded to the contracted state is also accompanied by the broadening of the amide I' mode of -helices.The full width at half maximum (FWHM) of this mode increased from 40 ± 4 to 48 ± 6 cm −1 , indicating that a broad population of hydrogen bonds with different bond strengths exists in the contracted forisomes adsorbed on the gold surface.The observed changes of the amide I' band of -helices indicate a weakening of a native network of hydrogen bonds during the uptake of Ca 2+ and transition to the contracted state.This may be accompanied by some rearrangements of helices (consistent with integral intensity change) leading to the formation of hydrogen bonds to water, which forisomes takes up during the transition to the contracted, state.
Note that the amide I' band of forisomes overlaps with a strong  as (COO − ) mode at 1596 cm −1 (see gray areas in Figure 2a,c).In the absence of Ca 2+ ions, the corresponding  s (COO − ) mode is centered at 1410 cm −1 .In the presence of Ca 2+ ions, the mode at 1410 cm −1 disappears from the PM IRRA spectrum while the intensity of the  as (COO − ) stretching mode at 1596 cm −1 decreases (Figure 2b).Depending on the cation present with the EDTA 2− anion, its spectrum shows the  as (COO − ) stretching mode in the range 1590−1610 cm −1 and the  s (COO − ) mode in the range 1400−1410 cm −1 . [24]Therefore, the strong  s (COO − ) mode at 1410 cm −1 and the  as (COO − ) at 1596 cm −1 are assigned to the EDTA 2− anion co-adsorbed with or within forisomes on the gold surface.Except for EDTA, Asp and Glu in forisomes give rise to the  as (COO − ) and  s (COO − ) stretching bands. [15,25]The wavenumber of the absorption maxima of these modes in the presence and absence of Ca 2+ in the three successive reaction cycles are shown in Figure 3.The  as (COO − ) modes at 1595 and 1540 cm −1 are observed in the PM IRRA spectra in Figure 2a-c in both, Ca 2+ -free and Ca 2+ -containing solutions (see also Figure 3).The  s (COO − ) stretching mode appears at 1367−1369 cm −1 .In the presence of Ca 2+ , the position of the  s (COO − ) mode is upshifted by ≈30 to 1396 cm −1 .
Nara et al. [26,27] predicted the characteristic (C═O) stretching frequencies in coordination complexes of the acetate anion and Na + , Mg 2+ and Ca 2+ ions using the Hartree-Fock method.These calculations were carried out for relatively small model systems and provide a basis for the analysis of the interactions between divalent cations and protein carboxylate groups.The coordination of divalent cations to the carboxylate residue has a characteristic influence on the position of the absorption maximum of the  as (COO − ) and  s (COO − ) bands as summarized in Table S2 (Supporting Information).Briefly, a free carboxylate group gives the  as (COO − ) mode in the 1550−1575 cm −1 range and the  s (COO − ) in the 1360−1380 cm −1 range (Table S2, Supporting Information). [26]These values correspond well to the positions of the  as (COO − ) and  s (COO − ) IR bands of the expanded forisomes in the absence of Ca 2+ in the electrolyte solution (Figure 3).
As shown in Table S2 (Supporting Information) for a carboxylate group with a bidentate bridging and pseudo-bridging (with D 2 O) coordination to Ca 2 , the  as (COO − ) signal is expected in the 1570−1590 cm −1 range, while the  s (COO − ) signal is expected in the 1380−1400 cm −1 range. [26,28]In a Ca 2+ -containing electrolyte solution, in contracted forisomes the  as (COO − ) absorption mode has two components, at 1595 and 1540 cm −1 , while the  s (COO − ) mode is found at 1396 cm −1 (Figure 3).PM IRRAS results indicate that the carboxylate groups in the side chains of Asp and Glu in forisomes bind Ca 2+ in a bidentate bridged and/or pseudobridged coordination.
Figure 3, Figures S4 and S5 (Supporting Information) show the results of the analysis of the spectral changes occurring in three successive reaction cycles.Immersion of the forisome film in the expanded state into CaCl 2 solution for 15 min causes spectral changes analogue to those shown in Figure 2b.The recovery of the native expanded state required a significantly longer time.At the beginning of the experiment, 60 min were sufficient to record a spectrum characteristic for expanded forisomes exemplified by Figure 2a,c.Over the 3 days of the experiment, the recovery time increased suggesting a gradual loss of the forisome activity.
As concluded from the IR spectra, adsorbed forisomes retain their native function and react to Ca 2+ ions by contraction.The forisome film is heterogeneous in structure.Thus, individual forisomes or layers of forisomes may interact differently with Ca 2+ and they may have a different orientation.Scanning electrochemical microscopy (SECM) in the feedback mode is a superb method to probe the heterogeneity and activity of adsorbed proteins on a micrometer length scale. [29]The schematic setup of a SECM experiment for the analysis of forisome layers is shown in Figure 4a.A microelectrode (ME) with a diameter of 25 μm (radius r T = 12.5 μm) is brought to a distance of 10 μm above the forisome-covered gold surface by recording an approach curve.The relatively large ME was selected in order to enable a larger working distance, which is in the range of the r T .A working distance of 10 μm was selected to minimize mechanical contact of the ME with protruding micrometer-sized protein aggregates.A larger ME also provides a higher sensitivity for slow kinetics of mediator regeneration at the surface of adsorbate-covered gold substrate.For an illustration of this methodical aspect, see Figure 4 of Ref. [30] The working solution is supplemented with a redox mediator, here 1 mm K 4 [Fe(CN) 6 ], which is oxidized at the microelectrode under diffusion-controlled conditions: The electrolysis current at the ME at a given horizontal (x, y) coordinate is the signal, from which the images in Figure 4b-d were constructed.The ME current depends on whether and at which rate the oxidized form of the mediator ([Fe(CN) 6 ] 3-) is reduced at the gold surface.This reduction is spontaneously driven at the open-circuit potential of the gold surface that is adjusted to the bulk concentration of the electrolyte by the Nernst equation (see Figure 8 of Ref. [31]).While there is a constant driving force for the reduction of the mediator at the gold substrate, the adsorbed forisome layer inhibits the electron transfer reaction at the underlaying gold surface.The degree of inhibition is expected to depend on the local permeability of the forisome for the charged redox mediator ([Fe(CN) 6 ] 4-/[Fe(CN) 6 ] 3-) and on the local thickness of the forisome layer.Thus, a plot of the local microelectrode current is indicative on the permeability of the film toward the mediator and on its thickness.Thus, the measured current at the ME provides contactless, in-situ information about the local film properties.
The SECM experiments were recorded above a film obtained by adsorption of forisomes onto the gold electrode surface from a NaCl-containing forisome solution.The SECM image in Figure 4b shows a current plateau.A slight increase in the current toward the rear part of the image is due to a slight tilt of the scanning plane and the sample surface.The alignment of the sample is challenging due to the locally different response of the heterogeneous protein film.Superimposed on the image are circular regions with diameters of ≈50-60 μm in which the current is either decreased or increased compared to the surrounding regions (Figure S6, Supporting Information).In Figure 4b, there is a region with increased currents at y = 240 μm that does not appear in subsequent images.It may come from protein aggregates temporarily attached to the ME.To facilitate discussion, three spots, labeled A, B and C have been selected in Figure 4.For point B, the current profile along the y-axis are shown in Figure S6 (Supporting Information).The size and distribution of these regions agrees quite well with the size and distribution of the spots in the light microscopy images shown in Figure S2 (Supporting Information).The ME current above the spots is usually lower than over the rest of the surface indicating a slight blocking property of the forisome film at these locations.It is very interesting and also unexpected that the different forisome assemblies have a distinctly different effect on the SECM feedback current (point B in Figure 4).The reason for the current increase is unknown.Possibly, the local microstructure of the adsorbed forisomes may allow for the accumulation of the mediator ([Fe(CN) 6 ] 4-) in the protein, similar to EDTA 2− anions.A local increase in the mediator concentration in the forisomes will result in an increase in the measured current for the mediator oxidation.Combined identical location SECM-IRM, which is not available yet, would be necessary to prove this hypothesis.
The same region of the surface was imaged sequentially after the addition of Ca 2+ to the working solution (Figure 4c).Despite small changes in the measured current, features reflecting the forisomes can be identified at the same positions in Figure 4b and in Figure 4c.After the addition of Ca 2+ to the electrolyte solution, the size of spot B increased to a diameter of ≈80 μm (Figure S6, Supporting Information).An increase in the area of the spot is in line with a size change by forisomes interacting with Ca 2+ . [2,3]fter addition of Na 2 EDTA (to complex Ca 2+ ), further changes were observed in the SECM image of the same region as shown in Figure 4d: Spots A and C disappeared indicating desorption of forisomes from the gold surface.This observation agrees well with the IR studies described above and is consistent with the notion that water uptake, forisomes contraction and the associated volume increase of forisomes weakens the adherence of the protein to the gold surface.In Figure 4b there is a region with increased currents at y = 240 μm that does not appear in subsequent images.It may come from protein aggregates temporarily attached to the ME and acting similar to a mediator-loaded hydrogel.
Point B in Figure 4b-d shows a typical functional behavior of a forisome film: In the absence of Ca 2+ , the diameter is close to 50-60 μm (Figure 4b,d; Figure S6, Supporting Information) while in the presence of Ca 2+ the size increased to circa 90 μm (Figure 4c; Figure S6, Supporting Information).The SECM and ATR imaging of adsorbed forisomes give similar sizes of heterogenous assemblies form the adsorbed forisomes, which are much larger than expected for a single forisome grown in yeasts (ca.0.7 μm × 4.6 μm).Therefore, we conclude that a few bodies adsorb together on the gold surface forming agglomerates of condensed protein film.A statistical analysis including more spots (labeled as A to I in Figure 4b-d) is provided in Section SI-S7 and Figure S7 (Supporting Information).The response of the forisomes identified in Figure 4 is not uniform when the concentration of Ca 2+ is changed.Some, but not all, agglomerates maintain their biological activity after adsorption to the gold surface.The reason for this non-uniform behavior of adsorbed forisome bodies is unclear at present, but is also reflected by the locally different IR spectra recorded on the adsorbed forisome layers.The different orientation of forsiomes with respect to the substrate may play a major role in the observed heterogeneity in the response, but requires further investigation.

Conclusion
The results of this work clearly show that the giant forisomes, whose size is comparable to that of biological cells, adsorb on the gold surface.We found that the conformation and secondary structure depend on the composition of the electrolyte solution from which the forisomes were assembled.The -helices are the most abundant structural elements in forisomes.
Our results show that adsorbed forisomes retain their native response toward Ca 2+ .IR spectroscopy uncovered alternations on the protein film at the secondary and tertiary structural elements upon uptake/removal of Ca 2+ ions.Briefly, the secondary structure elements do not change between the expanded and contracted states.In the measured PM IRRA spectra, the asymmetric and symmetric carboxylate stretching modes in Asp and Glu reversibly change their position and intensity in the presence (contracted state) and absence (expanded state) of Ca 2+ .The measured spectral changes indicate a bidentate bridged and /or pseudobridged coordination of Ca 2+ and underline the involvement of acidic amino acids in the reaction cycle of forisomes.The activation of forisomes is expressed by 3D rearrangements of the protein bodies that "open" the mechanoprotein for the binding of Ca 2+ ions to Asp and Glu.A simultaneous change in the hydrogen bonding network of -helices could be caused by the uptake of water by forisomes during contraction as proposed by Groscurth at al. [9] The reaction of forisomes appears to be controlled by coordination of Ca 2+ ions to side carboxylate residues in Asp and Glu that reorients the corresponding protein fragments causing changes in the protein tertiary structure.Indeed, no Ca 2+ binding motif has been identified in forisomes, [17] strengthening the argument of structural rearrangements inside the protein allowing for inter-and intramolecular coordination of Ca 2+ ions.
IR microscopy and SECM experiments demonstrated that forisome films are heterogenous and some adsorbed protein aggregates retain their native function.Upon Ca 2+ uptake, the spotdiameter increased from 50 μm to ≈80 μm, and upon removal of Ca 2+ ions by complexation with EDTA, their diameter decreased to the initial value.These sizes are larger than the size of a single forisome indicating that several bodies agglomerate on the surface.
In the adsorbed state, the forisome contraction occurred immediately after exposure to Ca 2+ .In the first contractionexpansion reaction a fraction of forisomes, probably weakly associated with the surface, desorbed from the gold surface.The contraction of forisomes is connected to a volume increase and requires an increase in the area per protein body.This may lead to the mechanical removal of some weakly associated protein bodies from the substrate surface.Forisomes leaving the surface provide space for the strongly adsorbed, swelling protein bodies.The expansion process of adsorbed forisomes is slow and was prolonged in subsequent contraction-expansion cycles to several hours.This indicates a gradual loss of the forisome activity.
Interestingly, EDTA 2− and possibly [Fe(CN) 6 ] 4-anions have an ability to accumulate within the adsorbed forisomes.The accumulation of small molecules inside of the forisomes could be responsible for the prolonged expansion time.In addition, over the long duration of the experiment, unavoidable contact of the forisome film to O 2 may slow down the reaction.The ability of forisomes to accumulate small organic molecules in their expanded state possesses an application potential.For example, they could be used as carriers of small organic molecules that could be released by triggering the longitudinal contraction of forisomes by physiological (Ca 2+ ) or even non-physiological triggers.
Summarizing, we demonstrate that adsorbed forisomes retain their native physiological function.This work contributes to the understanding of structural changes, at the submolecular level, that are responsible for the function of the entire forisomes opening new possibilities for biotechnological applications of this fascinating mechanoprotein.
Self-Assembly of MtSEO-F1 Forisome Bodies on Gold Surfaces: Microscope glass slides (VWR International BVBA, Belgium) were cut in pieces of 1.0 cm × 2.5 cm and rinsed with water and 2-propanol.Afterward, the glass slides were dried in an Ar stream.On the cleaned glass surface, layers of 3 nm Cr as adhesion promoter and 200 nm Au were evaporated using a Tectra MinCoater instrument (Tectra GmbH, Germany).Before protein self-assembly the slides were rinsed with water and ethanol, dried with Ar, and placed in an UV/ozone cleaner (Bioforce Nanoscience Inc., USA) for 10 min.
The artificial MtSEO-F1 forisomes were adsorbed on gold surfaces from aqueous solutions containing 10 μg mL −1 of the protein.The electrolyte solution was either 0.1 m KClO 4 + 0.01 m Na 2 EDTA or 0.1 m NaCl + 0.01 m Na 2 EDTA + 0.05 m NaHSO 3 + 10 μm CoCl 2 .NaHSO 3 (reductant) and CoCl 2 (catalyst) act as chemical oxygen scavenging system that was used because reversion of forsiomes to the longitudinally expanded state is adversely affected by dissolved O 2 in the solution by formation of disulfide bridges. [10]The solvent was either H 2 O or D 2 O.In the electrolyte solution containing KClO 4 the adsorption time was at least 12 h long while in NaCl 3 h were sufficient to adsorb a forisome film.After the adsorption, the protein film was rinsed with the corresponding electrolyte solution and stored for 1 h in 0.01 m Na 2 EDTA, 0.05 m NaHSO 3 , and 10 μm CoCl 2 in water to remove weakly adsorbed protein.
PM IRRAS: PM IRRA spectra were measured with a Vertex 70 spectrometer and an external reflection setup (Bruker, Germany) containing a photoelastic modulator with the frequency of 50 kHz and a demodulator PMA 50 (Hinds Instruments, USA).The IR spectra were collected in dry air atmosphere.All spectra were recorded with a resolution 4 cm −1 .The maximum PEM efficiency was set for the half-wave retardation at 1600 cm −1 for the analysis of the amide I' and II' bands.Each spectrum represents 800 averaged spectra.The angle of the incident IR light was 80°.The PM IRRA spectra were processed using the OPUS v5.5 software (Bruker, Ettlingen, Germany).First, PM IRRA spectra of freshly adsorbed protein films at the gold|air interface were measured.In order to test the protein activity, the forisome films were immersed for 15 min into D 2 O electrolyte solution containing 0.1 m CaCl 2 + 0.05 m NaHSO 3 + 10 μm CoCl 2 and the PM IRRA spectra were measured in air after removal of the samples from the electrolyte solution.Afterward, the protein film was immersed in 0.1 m NaCl + 0.01 m Na 2 EDTA + 0.05 m NaHSO 3 + 10 μm CoCl 2 at least for 1 h.PM IRRA spectra of the protein film in air were measured.These two steps were repeated three times over ca.70 h.The fitting of the IR spectra is described in Section SI-S2 (Supporting Information).
IR Microscopy: IRM experiments were performed on a LUMOS apparatus equipped with a mercury-cadmium-telluride detector, circular apertures, Schwarzschild objective and motorized state for collection of the IR spectra from selected, using optical microscope, areas of the sample.The IR spectra were collected in attenuated total reflection mode (ATR IRS) using a Ge crystal.The background spectrum was taken from an unmodified gold substrate.The ATR IR spectra were acquired at the gold|air interface in the spectral range 4000 -600 cm −1 from 10 μm × 10 μm regions of the surface in single reflection mode.The spectral resolution was 4 cm −1 .Each spectrum represents 80 averaged spectra.
SECM: Home-made SECM setup and electrochemical cell were used in these experiments.Briefly, the SECM setup contained a PG10 potentiostat (Jaissle Elektronik, Germany), a positioning system (Märzhäuser, Germany), a motor-controlled table for the correction of tilt of the cell (Zaber, Canada).The analog signal was converted into a digital using DAS1602/16 ADDA card.The details of the setup have been described before. [32]Pt microelectode with a diameter of 25 μm was used as a working electrode (WE).It was prepared by sealing a Pt wire in a glass capillary and exposing the cross-section of the wire by grinding and polishing.The ratio R g between the diameter of the glass sheath and the active electrode area was 9.64.Before use, the microelectrode was polished with a suspension of 0.05 μm Al 2 O 3 powder in water and finally rinsed with water.An Ag wire was used as a quasi-reference electrode and Pt wire as an auxiliary electrode.The approach curves and the SECM images were recorded in 1 mm K 4 [Fe(CN) 6 ] + 0.01 m Na 2 EDTA +0.1 m NaCl aqueous solutions.The potential of the microelectrode was set to 0.4 V versus RE for the oxidation of

Figure 1 .
Figure 1.PM IRRA spectra (upper panels) and their second derivatives (lower panels) of forisome bodies adsorbed from 10 μg mL −1 protein solution from a) 0.1 m KClO 4 + 0.01 m Na 2 EDTA in D 2 O solution for 20 h and b) 0.1 m NaCl + 0.01 m Na 2 EDTA, 0.05 m NaHSO 3 and 10 μm CoCl 2 in D 2 O solution for 2 h.Spectra were recorded at the gold|air interface after removal of the gold substrate from a given electrolyte solution.The absorbance is shown in arbitrary units.

Figure 2 .
Figure 2. Deconvoluted PM IRRA spectra of a) a freshly adsorbed forisome film from 10 μg mL −1 MtSEO-F1 + 0.1 m NaCl + 0.01 m Na 2 EDTA + 0.05 m NaHSO 3 + 10 μm CaCl 2 in D 2 O, b) after 15 min immersion of the same film in 0.01 m CaCl 2 + 0.05 m NaHSO 3 + 10 μm CaCl 2 and c) after further 30 min immersion of sample (b) in 0.1 m NaCl + 0.01 m Na 2 EDTA + 0.05 m NaHSO 3 + 10 μM CaCl 2 .Spectra were recorded at the gold|air interface after removal of the gold substrate from a given electrolyte solution.The experimental PM IRRA spectra are labeled with blue squares and the overall fitted spectrum with a black line.Red lines and fields correspond to the amide I' modes, cyan lines and fields -the COO − stretching modes in the protein, gray lines and gray-cyan fields -the COO − stretching modes in the protein and DETA, gray lines and fields -the COO − stretching modes in EDTA.The absorbance is shown in arbitrary units.

Figure 3 .
Figure 3. Positions of  as (COO − ) (squares) and  s (COO − ) (circles) stretching modes in side chains of Asp and Glu amino acids in forisomes (cyan) and in EDTA/forisome (gray) adsorbed on the gold surface during the reaction cycles after subsequent addition of Ca 2+ ions and their complexation by EDTA ("No Ca 2+ "), top abscissa shows time of the experiment at which the spectra were measured.

Figure 4 .
Figure 4. SECM feedback imaging of adsorbed forisome layers on gold.a) Schematics of the working mode (cross-section, not to scale!); b-d) experimental SECM images of forisome layers on gold adsorbed from 10 μg mL -1 concentration of forisome + 0.01 m NaCl and 0.01 m Na 2 EDTA solution for 60 h.The panels b to d) show sequential images of an identical region recorded with a Pt microelectrode, r T = 12.5 μm, at E T = 0.4 V versus Ag RE, translation rate v T = 7.5 μm s −1 , in 1 mm K 4 [Fe(CN) 6 ] + 0.1 m NaCl at a distance d = 10 μm between the microelectrode and the forisomecoated Au surface.The color scale highlights the deviation to the local background current.Specific conditions b) Na 2 EDTA, c) addition of 20 μL of 0.1 m CaCl 2 and d) addition of 40 μL 0.1 m Na 2 EDTA solution.