Design of Aqueous-Liquid Crystal Interfaces To Monitor Protein

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C: Surfaces, Interfaces, Porous Materials, and Catalysis

Design of Aqueous-Liquid Crystal Interfaces to Monitor Protein Aggregation at Nanomolar Concentrations Ipsita Pani, Hema M. Swasthi, Samrat Mukhopadhyay, and Santanu Kumar Pal J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b10863 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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The Journal of Physical Chemistry

Design of Aqueous-Liquid Crystal Interfaces to Monitor Protein Aggregation at Nanomolar Concentrations Ipsita Pani,†,§ Hema M. Swasthi,†,¶,§ Samrat Mukhopadhyay†,‡,¶,* and Santanu Kumar Pal†* †

Department of Chemical Sciences, ‡Department of Biological Sciences, and ¶Centre for Protein Science, Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, Mohali 140306, Punjab, India

ABSTRACT Amyloids are proteinaceous aggregates, the deposition of which is associated with neurodegenerative diseases such as Alzheimer’s disease. In vitro protein aggregation requires high protein concentration which is generally far from physiological concentration. Here, we utilize the interfacial properties of liquid crystals (LC) to monitor the membrane-induced aggregation of a bacterial functional amyloid, curli at nanomolar concentration. The binding event triggers an orientational transition of the LC which is accompanied by the appearance of dynamic spatial patterns enabling sensitive detection of LPS-mediated protein aggregation. Quantification of LC response shows a sigmoidal time profile, typical of a protein fibrillation assay. Curli is composed of two subunits (CsgA and CsgB) and is expressed on the outer membrane of Gram-negative bacteria containing lipopolysaccharides (LPS) endotoxin. CsgA forms the major subunit of curli, which is nucleated by the membrane-tethered minor subunit

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CsgB. Using an array of complementary tools, such as polarizing optical microscopy, fluorescence and atomic force microscopy imaging, we found that the patterned orientation of the LC in response to the binding of curli subunits with LPS corresponds to amyloid fibril formation. Furthermore, using the curli amyloid system, we have successfully demonstrated that membrane decorated interfaces of LC can be used to study heterotypic cross-seeding in amyloidogenesis. We believe that the LC-based system could be used as a probe to monitor mechanistic details of lipid-induced protein aggregation in the low concentration regime.

INTRODUCTION Amyloids are misfolded protein aggregates that share a common architecture comprising a cross-β motif.1 Deposition of amyloids is implicated in many devastating neurodegenerative diseases such as Alzheimer’s, Parkinson’s diseases and prion-based encephalopathies.1,2,3 Many disease-associated amyloid forming proteins such as Aβ, α-synuclein and human prion are known to interact with membrane.4 A typical in vitro protein aggregation requires high protein concentration which is generally far from physiological concentration. For instance, Aβ whose aggregation is implicated in Alzheimer's disease is present in nanomolar concentration under physiological condition. However, the critical concentration of Aβ required for spontaneous in vitro aggregation is in micromolar concentration.5 Thioflavin T (ThT), a widely used fluorescent probe to monitor in vitro protein aggregation, fails to trace the early amyloid oligomers that is known to be toxic than the final aggregates.6 Additionally, the commonly used biophysical techniques such as NMR, FTIR to study protein aggregation require high protein concentration. Therefore, a sensitive and inexpensive technique is essential to have a better understanding of the oligomerization and to monitor protein aggregation in the low concentration regime.


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Liquid crystals (LC) possess a very low interfacial energy and their ordering transition is known to be influenced due to adsorbate-induced changes in the interfacial energy of the LC. Perturbation occurring at LC- aqueous interface, for instance, adsorption of targeted species can trigger a macroscopic rearrangement of the LC that are a distance of 1-100 µm from the interface.7 The change in the director configuration of LCs due to interfacial events that disrupt the orientation of the LC can be monitored by polarized optical microscopy. Owing to strong optical anisotropy of the LC and highly environment sensitive alignment, they have been exploited in a panoply of sensing applications, which includes proteins, lipids, DNA, endotoxin, etc.8-13 Aqueous-LC interfaces have also emerged as biomimetic systems for the study of complex biomolecular interactions. For example, we reported binding of proteins and different cell wall components to LPS, which led to changes in orientational ordering transition of the LC at those interfaces.14,15 Recently, LCs have been shown to exhibit unique responses towards peptides that aggregate at the membrane interfaces.16 It was found that ordering patterns in LC films strongly depend on the secondary structures of the proteins at the aqueous interface.16, 17 Using infrared-visible sum-frequency spectroscopy, Kim et al have shown a strong correlation between the LC domains observed and the oligopeptide secondary structure.17 Proteins with a secondary structure rich in β-sheet content showed dendritic patterns on lipid-decorated interfaces of the LC.16, 17 Therefore, we asked if it is possible to exploit the interfacial properties of the LC to monitor the mechanistic details of protein aggregation at nanomolar concentration. In order to answer this, we have used bacterial functional amyloid curli as a model system. Curli is expressed at the outer surface of Gram-negative bacteria such as Escherichia coli and Salmonella. The functions of curli include biofilm formation, cell-cell adhesion and hostpathogen interactions.18-20 Biofilm in the host cell can trigger a series of lethal infectious diseases


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like sepsis, urinary tract infection and chronic otitis media.21, 22 Disease-associated amyloids are formed due to the uncontrolled conversion of soluble form of a protein into its aggregated form. However, curli has regulated machinery for its assembly. Structural component of curli is composed of two subunits, CsgA and CsgB, and these subunits are secreted to the outer membrane as intrinsically disordered proteins (IDPs) (Figure S1). The mature curli subunits comprises N22 domain and five imperfect repeats. CsgA and CsgB have molecular weight of ~ 13 kDa and they share 30% sequence identity. The accessory proteins maintain solubility and assist the translocation of curli subunits to the bacterial outer surface.23 On the surface, membrane anchored CsgB nucleates the aggregation of CsgA.23-25 The outer leaflet of gramnegative bacteria is made up of lipopolysaccharide (LPS) also known as endotoxin. LPS is composed of a negatively charged Lipid A, core oligosaccharides and O-antigen.26 Previously, we have shown that the electrostatic interaction of CsgB/CsgBt (19-amino acids are deleted from the C-terminal of CsgB) with anionic detergent or LPS induces spontaneous oligomerization in the protein and these oligomers efficiently nucleate the aggregation of CsgA.27 We would like to state here that CsgBt shows aggregation behavior similar to the full length CsgB and is easier to handle.27 The aggregation studies were performed using conventional biophysical techniques which required µM protein concentrations. In this work, we sought to investigate aggregation behaviors of CsgA and CsgBt at LPS-laden aqueous interfaces of the LC at low concentration. MATERIALS AND METHODS Materials LPS (from Escherichia coli 0111:B4), Tris buffered saline (pH=7.4), and DMOAP (dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride) were purchased from SigmaAldrich (St. Louis, MO). Sulfuric acid, hydrogen peroxide (30% w/v) and the 5CB LC were purchased from Merck. Ethanol was obtained from Jebsen & Jenssen GmbH and Co., Germany


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(Sd. fine-chem limited). Milli-Q water (Millipore, Bedford, MA). Fischer’s Finest Premium Grade glass microscopic slides were purchased from Fischer Scientific (Pittsburgh, PA). Gold specimen grids (20 µm thick, 50 µm wide, 283 µm grid spacing) were purchased from Electron Microscopy Sciences (Fort Washington, PA). Isopropyl β-thiogalactopyranoside (IPTG) was obtained from Gold Biocom (USA), Guanidine hydrochloride (GdnHCl) was purchased from Amersco and 30 kDa filters was obtained Merck Millipore. Cleaning of Glass Substrates and DMOAP-decoration The glass microscope slides were cleaned with freshly prepared piranha solution (70:30 (v/v %) H2SO4:H2O2) and kept for 1 hour at 80 °C. The glass slides were then rinsed with copious amount of Milli-Q water, dried under a stream of nitrogen gas and kept in oven at 100 °C for at least 3 h prior to use. Cleaned glass slides were then immersed in an aqueous solution containing 0.1 % (v/v) DMOAP solutions in DI water for 30 min at room temperature. The DMOAP-coated glass slides were then rinsed with DI water to remove unreacted DMOAP from the surface, dried under a stream of nitrogen gas and kept in oven for 6 h. Preparation of LC films in TEM grids Gold grids were placed on DMOAP-coated glass slides. The grids were filled with approximately 0.3µL of 5CB and the excess LC was removed with the help of a syringe to produce a film of uniform thickness. Formation of Self-Assembled Layers of LPS Powdered LPS was dissolved in Milli-Q water at room temperature to obtain 80 μg/mL concentration. The resulting solution was sonicated for 15 min and vortexed for 5 min at room temperature. The LC filled gold grid was then contacted with LPS solution in an optical cell and incubated for 2 hours. The LPS laden interface was then washed twice with Tris buffer (20 mM pH 7.4) to remove the excess LPS prior to the addition of protein.


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Optical Characterization of LC films The optical appearance of the LC film immersed in 20 mM Tris buffer pH 7.4 was observed by using a polarizing optical microscope (Ziess Scope.A1) in transmission mode. Each image was captured with a Q-imaging camera mounted on the microscope with an exposure time of 80 ms. Fluorescence imaging was performed with a Ziess (Scope A1) fluorescence microscope. All the experiments have been repeated at least thrice. At a particular time point, we have captured the response of four grid squares to show average response of the grid. In the figures, we have shown only one representative grid square to get a clear picture of the progression of aggregation. Protein expression and purification CsgA and CsgBt were expressed and purified as described previously.27 Briefly, CsgA and CsgBt were overexpressed using 500 μM and 250 μM IPTG, respectively. Induced cell pellet from 500 mL culture was lysed in 50 mL of 8 M GdnHCl in 50 mM potassium phosphate buffer pH 7.3 and stirred for 24 h. Lysate was centrifuged and the supernatant was allowed to bind with Ni-NTA for an hour at room temperature. The protein of interest was eluted using 50 mM potassium phosphate, 125 mM imidazole, pH 7.3. The eluent was passed through 30 kDa filter and the filtrate was buffer exchanged with 50 mM potassium phosphate buffer pH 7.4. The protein concentrations were determined using ε280 of 10810 M-1cm1

, 7680 M-1cm-1 for CsgA and CsgBt, respectively. The purified proteins were immediately

diluted to either 500 nM or 1 μM in chilled 50 mM potassium phosphate buffer pH 7.4 for further use. AFM Imaging CsgBt (100 nM, 20 mM Tris pH 7.4, 3 mL) was incubated on the LPSdecorated LC gold grid for 3 h. The gold grid was taken out of reaction mixture and 100 μL of buffer 20 mM Tris pH 7.4 was added to the grid and sample was collected thoroughly from the gold grid disturbing the LC layer. Ten μL sample was placed on a freshly cleaved and water-


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washed mica (Grade V-4 mica from SPI, PA). After 10 min the mica was washed thrice with 100 μL of milli-Q water and allowed to dry under nitrogen stream for 15 min. The AFM images were taken within 1 hour after the saturation of protein at the aqueous-LC interface. Tapping mode in air was performed on Innova Bruker AFM using antimony-coated silicon tip of 8 nm radius and the images were processed using WSxM software.28 Quantification of LC response The grayscale intensity of the polarized optical micrographs was measured by ImageJ software. The mean grayscale intensity for each optical micrograph was taken as an average of the intensity of four different grid squares. The overall variations in the mean grayscale intensity over different sets of experiments were observed to be 5-15 %. RESULTS AND DISCUSSION Our first set of experiments employed polarized optical microscopy (POM) to investigate the ordering of LC upon interaction of CsgBt at LPS-laden aqueous-LC interface. Figure 1a shows the optical photomicrograph of nematic 5CB (4-Cyano-4'-pentylbiphenyl) LC confined within a TEM gold grid supported on a DMOAP coated glass slide in contact with an aqueous dispersion of 80 µg/mL LPS in 20 mM Tris buffer (pH=7.4) solution. As reported previously, 5CB aligns homeotropically in presence of LPS and exhibits a dark optical appearance under crossed polars (Figure 1a and 1c).9,

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Adsorption of proteins onto lipid decorated LC

interfaces is known to trigger an ordering transition in LC from dark to bright.13, 17 This transition can be mediated by a range of underlying phenomena like disruption of membrane integrity by penetration of proteins into the lipid layer, hydrolysis of the lipid layer by enzymatic activity of peptides, protein-membrane interactions and so on.13, 29 Upon addition of 100 nM concentration of CsgBt, we observed a bright optical appearance and formation of branch-like patterns throughout the LC image within seconds (Figure 1b and 1d).


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CsgB

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Figure 1. (a, b) Polarized light images (crossed polars) of 5CB LC hosted within TEM grids at LPS-laden aqueous-LC interface and after introduction of 100 nM of CsgBt into the aqueous phase, respectively. The change in the optical appearance of the LC is consistent with an ordering transition of the LC from (a) homeotropic (inset: conoscopic image confirming homeotropic orientation) to (b) planar/tilted orientation. (c, d) Schematic illustration of CsgBt induced responses of 5CB LC at LPS-laden aqueous-LC interfaces corresponding to (a) and (b), respectively. (e) A model illustrating CsgB mediated fibrillation of CsgA on the outer leaflet of Gram-negative bacteria. The scale bar is 50 µm.


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We hypothesized that CsgBt oligomers upon interaction with LPS emblem their structures on LC interface. We carried out two additional control experiments to establish the role of LPS in the above-described response towards CsgBt. First, we observed that there was no change in optical appearance of the LC in contact with CsgBt without LPS at aqueous-LC interface upto 1 hour (Figure S2). Second, we studied the optical response of LC on the LPS decorated interface in the presence of a non-amyloidogenic protein e.g., BSA (10 nM) which showed a rapid ordering transition from dark to bright but, without any thread-like structural domains as observed with CsgBt (Figure S3). It should be noted here that the adsorption of BSA doesn’t bring about the fibrillar patterns in the LC interface. This is because the secondary structure of BSA is not rich in β sheet unlike amyloidogenic peptides. These results, when combined, provide evidence that the structural domains are due to the interactions of CsgBt with the LPS on the LC film. The schematic illustration of peptide induced responses with the LC film at aqueous interface is shown in Figure 1. As a next step, we sought to investigate the sensitivity of LPS-induced aggregation to CsgBt adsorption, for which we compared the dynamic response of the 5CB LC at different concentrations of protein in the aqueous phase. Figure 2 shows the optical response of 5CB upon exposure to various concentrations of CsgBt on the LPS-laden interface. It should be noted that in each case the concentration of LPS adsorbed onto the LC interface was 80 µg/mL, which is sufficient to retain the homeotropic anchoring of LC (Panel A in Figure 2). The results shown in Figure 2 (Panel B) demonstrate optical response of the LC (under crossed polars) upon introduction of 100, 50, 20 and 10 nM of CsgBt onto LC interface after 30 minutes of injection of the protein.


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10 nM

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Figure 2. Optical images (crossed polars) of 5CB LC hosted in TEM gold grid supported on DMOAP treated glass slides in contact with the LPS-laden aqueous interfaces followed by adsorption with varying concentrations of CsgBt. Panel A represents the optical micrographs of the LC aqueous interface after 2 hours of incubation with an aqueous solution of 80 µg/mL LPS in 20 mM of Tris buffer (pH 7.4). Panels B and C represent the crossed polars and bright field micrographs after the addition of 100 nM, 50 nM, 20 nM, 10 nM concentrations of CsgBt followed by incubation for 30 minutes on LPS-laden interface (corresponding to Panel A), respectively. The scale bar is 20 µm.


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Linear branch like bright domains were observed throughout the LC image upto 20 nM of protein which were also clearly visible in corresponding bright field micrographs (Panel C). However, no change in the optical appearance was observed at 10 nM of CsgBt suggesting insufficient interactions between LPS and CsgBt to induce ordering transition in LC. Previously, we have shown that LPS/negatively charged detergent induces rapid oligomerization of CsgBt, which in turn accelerates the aggregation of the protein.27 Hence, we conjectured that the thread-like structures formed at the aqueous-LC interface could be due to CsgBt amyloid fibrils. In order to understand and visualize the nanoscale morphology of these thread-like structures, we performed atomic force microscopy (AFM) experiments of the LPSladen LC film after adsorption of the protein. The AFM imaging revealed the formation of long and straight fibrils of ~7 nm height at aqueous-LC interface (Figure 3a). In general, amyloids form long and unbranched filaments with a height of 4-12 nm. However, we did not observe any fibrils when CsgBt was added onto LC interface without LPS decoration (Figure S4). Therefore, AFM images validate that the fibrillation of CsgBt on the interface is induced due to the presence LPS. Therefore, we concluded that the thread-like domains formed at those interfaces are CsgBt amyloid fibrils. Next, we thought to provide additional support to confirm the formation of fibrils using amyloid marker ThT binding assay at aqueous-LC interface. We observed that upon addition of 100 nM CsgBt and 2 µM ThT at LPS-laden LC interface, the optical appearance of the 5CB LC became bright without formation of any thread-like domains at that interface (Figure 4b). No such patterns were also visible even when the analyzer was removed (Figure 4c). We hypothesized that this might be due to competitive binding of ThT with LPS which prevents the anticipated ThT-amyloid interactions.


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Figure 3. (a) AFM image of CsgBt fibrils formed at LPS-laden aqueous-5CB interface and its corresponding height profile. (b,c) Polarized light images (crossed polars) of the POPC:POPG 3:1 (1 mg/mL) laden aqueous-LC interface of 5CB and after adsorption of 100 nM of CsgBt for 1 hour, respectively. (d) Bright field image after removal of the analyzer corresponding to (c). (e) The epifluorescence microscopy of ThT fluorescence after 7 hours of adsorption of 100 nM of CsgBt with 2 µM ThT. The scale bar is 20 µm. To validate this, we did two additional control experiments. First, we contacted 2 µM ThT directly at LPS-laden aqueous-LC interface without CsgBt. Interestingly, we observed an ordering transition of the LC from dark to bright (Figure 4h and i) similar to what was observed upon adsorption of 100 nM CsgBt and 2 µM ThT at those interfaces. This clearly established that strong interactions between LPS and ThT at the interface prevent the formation of branched structures in presence of CsgBt.


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Figure 4. Polarized optical micrographs of 5CB filled TEM grids. a) LPS laden LC interface after 2 hours of incubation b) polarized light (crossed polars) and c) bright field micrograph of LPS laden aqueous LC interface obtained 15 minutes after addition of 100 nM CsgBt and 2 µM ThT. d) polarized light micrograph after 2 hours of incubation with LPS e) crossed polars and f) bright field image after 15 minutes of addition of 100 nM CsgBt. g) polarized light micrograph after 2 hours of incubation with LPS h) crossed polars and i) bright field image after 15 minutes of addition of 2 µM ThT. Scale bar is 20 µm. Second, to verify that the thread like patterns are indeed amyloids fibrils, we performed experiments with other lipids (instead of LPS) which do not have direct interactions with ThT


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but can orient the LC homeotropically at aqueous-LC interfaces. For this, we first decorated the 5CB interface with a zwitterionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)

and a negatively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-

glycerol) (POPG) at 3:1 ratio attaining a final concentration of 1 mg/mL (dark appearance under crossed polars, Figure 3b). The interface was washed after an incubation of 2 hours, followed by adsorption of 100 nM CsgBt at the LC interface. Under polarized light, approximately 5 minutes after addition of CsgBt, the branch-like domains appeared which continued to grow until an equilibrium stage was realized after ~ 1 hour (Figure 3c). The bright field image also shows clear patterns of these aggregates (Figure 3d). At this stage, we performed ThT assay and found increased ThT fluorescence with time which confirms the formation of fibrils, corroborated with the AFM results. Figure 3e shows that epifluorescence microscopy images after adsorption of 100 nM CsgBt and 2µM ThT. In summary, these results provide evidence that LPS facilitates the aggregation of CsgBt into fibrillar structure that emblem into a LC interface and can be monitored by change in the optical appearance of the LC at aqueous-LC interface. Next, in order to gain additional insights into the early events of CsgBt-LPS interactions, we monitored the dynamic optical response of the LC to CsgBt adsorption as a function of time. Upon adsorption of CsgBt, we see that the dark appearance of the interface (in presence of LPS) slowly evolve into a bright texture with fibrillar patterns as shown in Figure S5 (1st row). For instance, upon addition of 100 nM CsgBt, LPS decorated LC interface started turning bright within ~ 5 minutes and there was a concomitant formation of spherical domains at the LC interface Figure S5 (1st row). The spherical domains transform into fibrillar structures associated with a change in the orientational transition of the LC from homeotropic to planar/tilted.


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Figure 5. Optical image of 5CB LC hosted in TEM gold grid supported on DMOAP treated glass slides in contact with the LPS-laden aqueous interfaces followed by adsorption with varying concentrations of CsgA. Panel A represents the optical micrographs of the LC aqueous interface after 2 hours of incubation with an aqueous solution of 80 µg/mL LPS in 20 mM of Tris buffer (pH 7.4) followed by washing excess free LPS from aqueous-LC interface. Panels B and C represent the crossed polars and bright field micrographs after the addition of 100 nM, 50 nM, and 20 nM concentrations of CsgA followed by incubation for 30 minutes on LPS-laden interface. The scale bar is 20 µm.


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The LC interface became bright with linear branch-like patches in ~15 minutes of incubation of the protein (Figure 5, 1st row). We measured the grayscale intensity of the interface at different time points to get insights into the extent of fibrillation at the aqueous LC interface. A plot of the mean grayscale intensity vs time shows a progressive increase in the brightness as a function of time. Additionally, we performed experiments with varying concentrations of CsgBt, ranging from 50-10 nM. We observed that the appearance of spherical domains at the interface depends on the protein concentrations. It is interesting to note that, as we increase the protein concentration, the interface turned completely bright at a faster rate. CsgB t at 50 nM and 20 nM exhibited a sigmoidal profile that is consistent with a typical protein fibrillation assay whereas, 10 nM CsgBt did not show significant change in the intensity. The grayscale intensity plot revealed that increase in the protein concentration induced a shortening in the lag phase of protein aggregation (Figure S6A). As reported earlier, electrostatic interaction of negatively charged detergent/LPS with C-terminal CsgBt is known to induce a rapid oligomerization in the protein.27 Thus we believe that the structural domains formed at the LC interface could be due to LPS-induced oligomers of CsgBt. LPS-induced oligomers of CsgBt can heteronucleate the polymerization of CsgA.27 Therefore, we speculated that the spherical domains formed at the LC interface due to CsgBt adsorption might also be capable of accelerating the aggregation of CsgA. Keeping this idea in mind, in the next experiment, we sought to investigate the following two things. First, we monitored the aggregation behavior of CsgA on LPS-laden LC interface and second, CsgBt induced fibrillation of CsgA at those interfaces. In order to test the proposition that CsgA can adsorb and interact with LPS, first we investigated the orientations of 5CB in the presence of 100 nM of CsgA at LPS-laden aqueousLC interface.


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Figure 6. a) Time lapse polarized light micrographs (crossed polars) of LPS-decorated LC interface in the presence of 20 nM CsgBt, 100 nM CsgA, and 20 nM:100 nM of CsgBt:CsgA. The scale bar is 20 µM. b) Mean grayscale intensity of optical micrographs of 5CB films as a function of time for 20 nM CsgBt, 100 nM CsgA, and 20 nM:100 nM of CsgBt:CsgA.


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Bright domains began to grow within 5 minutes and then full bright patterned structures were observed by 30 minutes (Figure 5, 1st column). However, upon addition of 20 nM CsgA, no change in the optical appearance was observed at the LC interface even after prolonged incubation of CsgA (Figure 5, 3rd column) suggesting no CsgA fibril formation. Figure S6B shows the grayscale intensity plot of time dependent aggregation of CsgA as a function of CsgA concentrations. These results indicate that LPS facilitates the aggregation of CsgBt at a much faster rate as compared to CsgA. In order to investigate whether LPS-induced oligomers of CsgBt can nucleate CsgA aggregation we next performed experiments using CsgA in presence of catalytic amount of CsgBt at LPS-LC interface. Figure 6 shows a plot of the mean grayscale intensity as a function of time for the individual proteins and the mixture. CsgA (100 nM) fibrillates and attains saturation in ~30 min. Interestingly, when a mixture of 20 nM CsgBt and 100 nM CsgA was added to the interface, bright elongated textures started appearing at 5 min which reached saturation within ~15 min. This observation suggests that sub-stoichiometric amount of CsgBt nucleates the aggregation of CsgA. Also, the intensity profile of the CsgA:CsgBt (100:20) mixture shows a faster fibrillation than 100 nM CsgA suggesting that CsgBt nucleates aggregation of CsgA. However, we do not rule out the possibility of co-aggregation since 100 nM CsgA itself aggregates in that condition. In another experiment, whereas, only 500 nM CsgA was able to switch to bright patterns of the LC at LPS-LC interface within 5 minutes, addition of 100 nM CsgBt:500 nM CsgA, a spontaneous change in the optical appearance was observed at those interfaces with bright threads and after that no further change in the LC interface (Figure S5). This observation suggests that the rapid change observed in the LC interface upon addition of CsgBt:CsgA (100


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nM:500 nM) might be due to LPS-induced oligomers of CsgBt which accelerate the aggregation of CsgA. CONCLUSIONS In this work, we have used aqueous liquid crystalline interfaces for studying the aggregation behavior of biofilm-forming amyloidogenic proteins. We demonstrate that the binding of CsgBt/CsgBt:CsgA to LPS induces an ordering transition from homeotropic to tilted state at LCaqueous interface and these patterned orientation of LC represents fibrillation of these intrinsically disordered proteins at those interfaces. Aggregation of curli subunits on the thin films of LC can be easily visualized by simple optical microscopy. Quantification of the LC response as a function of time suggested that under identical reaction conditions CsgBt aggregates fasters than CsgA. Further, the evaluation of LC response at various stages of the protein fibrillation indicated that the aggregation of CsgA might be nucleated by CsgBt on the LPS-LC interface. The detection limit of our LC based method towards CsgBt and CsgA was found to be ~20 nM and 50 nM (Figure 2 and 5), respectively. Using AFM imaging we confirmed that the dendritic species formed on the LC-LPS interface are amyloid fibrils. The results demonstrated here are comparable to the previous study

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that was carried out using

micromolar concentration. We believe that our work will lead to a promising approach to design LC-based system to study in vitro aggregation of proteins that are present at a low concentration under physiologically conditions. This LC-based technique can be used as a general method to detect membrane-induced early amyloid intermediates and also can be used to monitor protein aggregation process in the low protein concentration regime.

ASSOCIATED CONTENT


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AUTHOR INFORMATION Corresponding Author *[email protected] and [email protected] Author Contributions §

These authors contributed equally.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT S.K.P. is grateful for the financial support from INSA bearing Sanction No. SP/YSP/124/2015/433. S.M. acknowledges the DST Nano Mission and the MHRD, Govt. of India for financial support. I. P. and H.M.S. acknowledge fellowships from IISER Mohali. We thank Sumyra Sidiq for helping in epifluorescence experiments.

We sincerely thank Dr.

Sabyasachi Rakshit for many helpful discussions. Supporting Information Available Detailed sequence of proteins used in the study, POM and AFM images for the control experiments and quantification of aggregation of individual curli subunits (Fig. S1-S6). This information is available free of charge via the Internet at http://pubs.acs.org ABBREVIATIONS LPS, Lipopolysaccharide; LC, Liquid crystal; Aβ, Amyloid β


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TOC Graphic

CsgA/CsgBt + aqueous LPS

DMOAP coated glass LPS-H2O-LC

20 µm

20 µm

CsgA/ CsgBt

Au grid

Aqueous LPS solution

Au grid

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DMOAP coated glass CsgA/Bt-LPS-H2O-LC

1.6µm


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Design of Aqueous-Liquid Crystal Interfaces To Monitor Protein

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