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Chemically engineered synthetic lipid vesicles for sensing and visualization of protein - bilayer interactions Navneet Dogra, Rajesh Prabhu Balaraman, and Punit Kohli Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00366 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Bioconjugate Chemistry

Chemically engineered synthetic lipid vesicles for sensing and visualization of protein - bilayer interactions Navneet Dogra,1,2, 3,* Rajesh P Balaraman,1 and Punit Kohli1,* 1Department 2IBM

of Chemistry and Biochemistry, Southern Illinois University, Carbondale, IL 62901

T. J. Watson Research Center, Yorktown Heights, NY 10058, United States

3Department

of Genetics and Genomics Sciences, Icahn School of Medicine at Mount Sinai, New York

10029, United States. Contact information: *[email protected]; *[email protected] Keywords: vesicles, liposomes, membrane, FRET, biosensors, synthetic lipids

Table of content: Engineered vesicles for protein sensing

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

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ABSTRACT: From pathogen intrusion to immune response, cell membrane plays an important role in signal transduction. Such signals are important for cellular proliferation and survival. However, measurement of these subtle signals through the lipid membrane scaffold is challenging. We present a chromatic model membrane vesicle system engineered to covalently bind with lysine residues of protein molecules for investigation of cellular interactions and signaling. We discovered that different protein molecules induced differential spectroscopic signals, which is based on the chemical and physical properties of protein interacting at the vesicle surface. The observed chromatic response (CR) for bound protein molecules with higher molecular weight was much larger (~5-15X) than those for low molecular weight proteins. Through mass spectrometry (MS), we found that only six out of sixty (10%) lysine groups present in bovine serum albumin (BSA) were accessible to membrane of the vesicles. Finally, a “sphereshell” model representing protein-vesicle complex was used for evaluating the contribution of the van der Waals interactions between proteins and vesicle. Our analysis points toward contributions from van der Waals, hydrophobic, and electrostatic interactions toward observed CR signals resulting from molecular interactions at the vesicle membrane surface. Overall, this study provided a convenient, chromatic, semiquantitative way of detecting biomolecules and their interactions with model membranes at subnanomolar concentration.


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Signal

Protein A

Protein B Figure 1. An overview of present study. (From left to right) NHS labeled PDA vesicles covalently bind with the lysine residues of two different proteins (MWproteinA < MWproteinB). Depending upon their physicochemical properties, the protein molecules interact differently with vesicle surface. Proteins produce unique spectroscopic signals. MW represents molecular weight of protein molecules.

INTRODUCTION From exo- to endo-cytosis and pathogen intrusion to immune response, lipid bilayer plays an important role in signal transduction.1 For instance, lipid - protein interactions influence crucial cellular processes such as neuro-synaptic signaling,2 membrane fusion,3 Golgi vesicle genesis,4 and lipid-associated enzymatic reactions.5 During kinase based phosphorylation, a lipid micro-domain (lipid raft) induces protein recruitment process which leads to a cascade of signaling pathway.5 Similar processes have been found to occur during caveolae formation, glycosyl-phosphatidylinositol (GPI)-anchored 3 ACS Paragon Plus Environment

proteins


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sorting,6 and T-cell antigen receptor signaling.5 The prevailing understanding about lipid induced signaling points us toward subtle changes that occur in lipid micro-environment, which eventually leads to a signaling cascade.1, 7 Such processes are widely important and extremely interesting in molecular and cellular biology. However, measurement of these subtle transduction signals through the lipid membrane scaffold is challenging.7-9 Due to their complex membrane structure, individual protein interactions are challenging to investigate in cells. Simple cell free model membrane systems (synthetic vesicles) are frequently used to understand mechanism of complex cellular functions.7,

9-11

Vesicles with dimensions and composition similar to

cellular structures (1-20 μm) have been used as a model for cellular studies,

12, 13

and smaller synthetic

vesicles (50-100 nm) have been used to mimic synaptic vesicles and exosomes.3, 14, 15 Through similar systems, it has been shown that lipid membrane and biomolecular interactions have direct influence on membrane curvature, dynamics, permeability, and various mechanical properties.10,

11, 13

Furthermore,

cellular proliferation and death has also been investigated using BAX-mediated pore formation in vesicles.16 Recently, synthetic vesicle systems were used to investigate ligand-receptor interactions on lipid membrane.13,

17-20

This was achieved by incorporating a “lipid like molecule” called polymerized

diacetylenic acid (PDA) into the lipid membrane scaffold (Scheme 1).21, 22 The PDA molecules exhibit changes in the optical absorption and fluorescence emission spectra following application from external stimulations.23 The underlying principle of PDA signaling to molecular interactions is that, PDA backbone provides a transducing element in the vesicles for probing protein-membrane interactions.24


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Vesicle A, B, and C

Scheme 1. (A) Structure of chemicals synthesized to mimic lipid probes for vesicle preparation are, 1 (PDA), 1a (NHS-PDA), 3 (DMPC), 4 (Rhodamine-PDA). Vesicle A, B, and C were prepared using three different ratios of 1:1a:3. Giant vesicles were prepared using 1:1a:3: and 4, for fluorescence microscopic studies.

Previously, we have studied that glucose (receptor) molecules covalently-bound to the vesicles surface yield about three times larger CR response for E. Coli sensing as compared to non-covalently-bound glucose based sensor.25 The difference in observed spectroscopic response in these studies was attributed to larger stress transport efficiency for covalently bound sensors than those for the non-covalently bound sensors.26,

27

Using colorimetric properties of PDA for probing molecular interactions at membrane



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surface, we have extended our model membrane system to protein and bacterial sensing, and as antibacterial drug carriers using Fluorescence Resonance Energy Transfer (FRET) response.25, 26, 28 In this manuscript (Fig. 1), we have created NHS-PDA embedded cell free model membrane vesicles for investigation of multi-protein reaction at bilayer surface (essentially, any protein with accessible lysine groups can covalently bind at the membrane surface). Furthermore, we demonstrate monitoring of protein – protein, protein - bilayer, and protein - enzyme interactions at membrane surface. This study reveals that CR response following the covalent binding of protein molecules on the vesicle surface is dependent on physico-chemical properties of protein including shape, size, lysine residues, and charge of the molecules (Fig. 1).

We found that the CR signal for protein-vesicle system were further amplified by

>100% following enzymatic cleavage of protein molecules bound at the vesicles surface. The enhancement in CR signal after enzymatic protein reactions was also found to depend upon the molecular weight (MW) of proteins. The enzymatic cleavage of the larger protein (myosin-540 kDa and IgG-150 kDa) bound to vesicles yielded larger CR signals as compared to cleavage of the smaller protein molecules (lysozyme-14.4 kDa and BSA-66.5kDa). FRET and fluorescence anisotropy (FA) measurements confirmed proximity of covalently-bound protein molecules to the vesicle membrane. Infrared spectroscopy (IR) was employed to study conformational changes in the PDA membrane backbone and mass spectrometry (MS) analysis suggested that only six out of sixty (10%) lysine residues in BSA were accessible for the covalent reaction between the membrane and BSA. Finally, we used a “sphere-shell” particle model to evaluate the contribution of van der Waals interactions in protein-vesicle complexes toward observed CR signal. Although, the proposed model does not take into account the short-range molecular interactions, this provides semi-quantitative information interpreting close-ranged molecular interactions. The present studies aid us in enhancing our understanding of biochemical reactions and


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interactions on model membrane surfaces in biologically relevant cellular systems and offer opportunities for development of highly sensitive and selective biosensors. RESULTS AND DISCUSSIONS Design, synthesis and characterization of PDA and NHS-PDA vesicles. To investigate proteinmembrane interactions and in-situ covalent binding of proteins with vesicles, our first step was to design and prepare reactive N-hydroxysuccinimide-polydiacetylene acid (NHS-PDA) vesicles. NHS activated carboxylic groups are highly reactive to nucleophiles, including amine, ammonia, water, and thiols.29 To identify ideal NHS-PDA concentration in the membrane for biomolecular binding and hence providing reproducible CR signal, we prepared vesicles composed of PDA : NHS-PDA : DMPC in three different ratios - 8:1:1 (vesicles A), 4:4:2 (vesicles B), and 1:8:1 (vesicles C) (Scheme 1, and Methods for synthetic procedure). Total lipid concentration for all vesicles was kept at 1 mM. Rhodamine-PDA (1 μM) was added only for FRET and fluorescence microscopic studies. Characterization of NHS activated vesicles: Auto-hydrolysis of NHS groups attached at the vesicle surface may contribute to the overall CR signal (Scheme 1S), which can make accurate data interpretation relatively difficult. Our experiments show that the half-life of reaction between NHS and amine from lysine at physiological pH is ~ 1 to 10 minutes, which is >10 times faster than that of NHS-water reaction (~2 hours to days).30 NHS-PDA vesicles A were stable (25% when

rhodamine-

PDA conjugated backbone distance (d) is less than 3.36 nm. In our experiments, the rhodamine-BSA and conjugated vesicle pair showed a FRET efficiency (E) ~72 % (Fig. 2B). This large E in the present studies was due to large spectral overlap change (J) between excited-state donor (rhodamine) and ground-state acceptor (PDA) and close proximity of BSA to the vesicle particles confirming vesicle – protein complex formation (Fig. 2B). Vesicles without NHS did not show significant J changes under same experimental conditions. The estimated ∆J = !1 −

$%&

( × 100 for our experiments was ~75%, where Jin and Jfin values

$'%&

were 8 × 1011 and 1.4 × 1012 M-1cm-1nm4, respectively. Fluorescence anisotropic analyses: FA investigations aided us in evaluating covalent binding between NHS-vesicles and protein molecules. Assuming monodispersed vesicles of diameter of 100 nm with estimated molecular weight of ~410 x 106 g/mole suggested that the RMW (=Mvesicle/Mprotein) ranges between 9 ACS Paragon Plus Environment


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103 to 106 for our experiments. RMW is measure of relative size of vesicle to protein attached to the vesicles. Large RMW in our experiments indicated that vesicles would behave almost stationary in comparison to protein 4 kDa < MW T (thermal energy) for the blue-to-red colorimetric transition for PDA chains is reported in the literature.36, 37 Overall, as a result of the protein binding at the bilayer, the CR signals appear to depend both on the physical properties of the protein molecules and chemical interactions between protein and vesicles. Electrostatic, hydrophobic, and van der Waals (vdw) contributions to the CR signal It is important to understand the factors that may contribute to the observed CR signal due to proteinvesicle interactions. We consider short-range forces including electrostatic, hydrophobic, and van der Waals interactions to the observed CR-MW response in our experiments. The vesicles in our studies were composed of ~90% fatty acid polymerized monomers and 10% DMPC. The pKa of closely-packed fatty acids in membrane is ~7.2 which is ~2 pH units higher than pKa of dilute fatty acid solution.38 For example, pKa for stearic acid in a dilute solution is ~539, 40 but apparent pKa of stearic acid in the monoand bi-layers is ~7.241 and 7.840 respectively. This large pKa difference in bulk and close-packed of stearic acid is attributed to negative charged bilayer-aqueous interface leading to double-layer ions including protons near to the interface.39 The overall vesicle membrane surface in our system is expected to be close to neutral at pH 7 (DMPC is also neutral at pH=7) because the membrane is closely resembled closepacked fatty acids. The indiscriminant covalent binding of the protein molecules used in our studies irrespective of their charge (positive, negative, and neutral, please see below) points to lack of strong electrostatic interactions between protein and vesicles such that the protein molecules can approach vesicle-aqueous interface through diffusion mechanism without experiencing large electrostatic forces. Therefore, the electrostatic protein-vesicle interactions are less likely to have a significant effect during covalent binding process. In fact, the diffusion-based protein-vesicle collision is the first step in a



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potentially multi-steps process for the observed CR signal due to protein-vesicle complex formation in our experiments. We also note that charged protein-vesicle complexes also remained stable for many days. This enhanced stability of protein-vesicle complexes come from the acquired charge on the vesicle surface due to protein covalent binding at the surface.40 At the experimental pH=7, all the protein molecules were charged except anti-IgG (Tables 1 and 2S). For example, the estimated charge q on BSA (pI~4.9) and myosin (pI~5.3) were -10 and -36 per molecule at pH~7 respectively, whereas lysozyme (pI~11) possessed q~+8 per molecule (Tables 1 and 2S). The overall charge on the protein molecules were estimated at pH = 7 using an online tool Protein Calculator v3.4 (available at http://protcalc.sourceforge.net/). It is important to note that although estimated overall charge on the protein molecules is known, the local surface charge distribution on the protein under experimental conditions is less clearly defined. This is because the estimated charge for a protein molecule is based on the charge on the amino acid residues in the protein sequence along with the charge contributions from the end amino acid groups. However, the actual charge at the protein surface can be different from estimated charge depending upon local environment conditions. For example, if an amino acid is in proximity to a group of non-polar or highly charged side chains, the expected charge for the amino acid can be different from the expected charge based on local surface charge distribution.42 Further, the protein-vesicle covalent binding may also influence changes in the local environments of both protein and membrane CR signal. Therefore, although the overall estimated charge on the protein molecules is large (Table 1), its contribution to the observed CR signal due to formation of the protein-vesicle complex is not fully understood. As noted above, electrostatic charge at protein-vesicle appears to be less dominant because the protein molecules (irrespective of variable charges) covalently reacted with the vesicles. We, therefore, emphasize a qualitative significance of the electrostatic contribution toward observed CR signal.


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Following covalent reaction, the protein and vesicles were in close contact with one another. The close intimacy between the protein and vesicle yield van der Waals interactions (Evdw). We modeled vdw interactions for protein–vesicle complexes by assuming protein and vesicle particles as hard sphere and hard shell (Eq. 1, please see SI for more details).43

These calculations allowed us to gauge short-range

vdw contributions for protein-vesicle complexes.   2d d2  ( R )(R R )  A 2 2 2 1 2 E  ( 123 ) ( )  ( ) [ln( )]  2d d2 6  2d d2 vdw 2d d2 )  ( ) 4  ( )  ( ) 4  ( )  ( ) (  R R R R R R R R  1 2 2 1 2 2 1 2   R2   2(d  h) (d  h)2  ( R )( R R )  A 2 2 2 1 2 ( 123 ) ( )( ) [ln( )] 2(d  h) (d  h)2  6  2(d  h) (d  h)2 2(d  h) (d  h)2 )( RR ) 4( R )( RR ) 4( R )( R R )   ( R2 1 2 2 1 2 2 1 2   



R1>>h, d, R2, h ≈0.2 nm

Eq. 1

where R1, R2, d, and h are vesicle radius, radius of protein, protein-vesicle distance, and bilayer thickness of the vesicle respectively. A123=5.9x10-21 J (see SI) is estimated for Hamaker constant of protein and vesicle across water medium. R1, d, and h of 100 nm, 0.2 nm, and 4.8 nm were assumed for modeling studies. d≈0.2 nm was chosen because protein molecules were covalently bound at membrane surface of the vesicles presumably with the presence of a monolayer of water molecules between the protein and the membrane.44 h=4.8 nm was estimated using molecular mechanics energy-minimized monomer configuration (ChemBioOfficeUltra). The changes in h after polymerization were minor and were ignored in the vdw calculations. Using Eq. 1, the vdw interaction calculations for larger protein with vesicle complexes were found to be much larger than for the smaller protein-vesicle complexes (Table 1). For example, Rvdw-kT=(Evdw/Ethermal

energy)

is ~1, 2, and 4 for lysozyme-, BSA-, and IgG-vesicle complexes

respectively. Here Evdw and Ethermal energy are vdw and thermal energy interactions. Although the estimated Evdw for the small MW protein-vesicle is comparable to that of the thermal energy (≈T), the magnitude 17 ACS Paragon Plus Environment


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of vdw interactions for large protein-vesicle complexes is significantly larger than the thermal energy contribution. Therefore, the vdw interactions between larger MW protein and vesicles are likely to contribute to the observed CR-MW trend in our experiments. This model, however, underestimate the vdw interactions for protein-vesicle complexes because our model assumes both the protein and vesicle are hard particles. These over simplified estimations in the model, although useful, does not take into account that both the vesicles and protein molecules are soft in nature and can easily be deformed. The increase in the intimate protein-vesicle contact due to soft nature of protein and vesicle particles is likely to increase vdw magnitude. Since the contact between spherical particles is least of all geometries, the assumption of spherical particles in the model may also induce discrepancy in the estimated vdw interactions because vdw energy is a summation over volume and possesses 1/r2 dependence. For example, Y-shaped IgG can have higher intimate contact with vesicles than assumed spherical IgG in Eq. 1. Further, the cylindrical myosin can wrap around a spherical vesicle more than half the circumference of a 100 nm diameter vesicle may yield higher vdw interactions. Also, the presence of dielectric gradient across membrane-water interface influences the intimate protein-vesicle contact or may lead to insertion of the protein molecules into the membrane. For example, lysozyme is known to insert in the membranes (see next paragraph below for more details).45 The consequence of protein insertion in the membrane will be significantly larger vdw interactions between protein and vesicles than those estimated from hard sphere-hard shell model (Eq. 1). Finally, hydrogen-bonding between vesicles and protein molecules can also contribute to observed CR signal and cannot be ruled out in our experiments. Rvdw-kT~1 for lysozyme-vesicle complex points to insignificant vdw contribution to the observed CR signal. Lysozyme46, BSA47, and IgG47 are known to insert into vesicle membrane. Lysozyme from chicken possesses 147 amino acid residues, of which 55 amino acid residues are hydrophobic in nature.45, 47

That is, ~37% of all amino acid residues in lysozyme are hydrophobic in nature which provides a


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driving force for its insertion into the bilayer of the vesicles. Finally, the dielectric constant () gradient from aqueous solution in the bulk solution to hydrophobic within the interior of the membrane provides a driving force for the insertion of some protein molecules into the membrane.44 For example,  for bulk water, bilayer-water interface, and hydrophobic interior of the bilayer are ~78,48 ~33-36 for ~20 mM salt,47 and 4-1044. The lysozyme insertion in the membrane will result in an enhanced protein-vesicle vdw and hydrophobic interactions contributing to larger changes in the membrane packing and deformation in the alkyl side chains in the membrane. The changes in the membrane packing and conformational changes in the alkyl side chains are known to result in the blue-to-red PDA transition11 which is the basis for observed CR signal in the present case. Overall, estimated Rvdw-kT for lysozyme may not fully explain vdw contribution to the observed CR signal, and the larger observed CR signal than that expected from Eq. 1 for the lysozyme is attributed to changes in the membrane packing and conformations related to its insertion in the membrane. The partial or full insertion of lysozyme into vesicle membrane will lower its accessibility for enzyme cleavage may also help in explaining relatively small increase (~3%) in the CR signal after enzyme reaction (Fig. 5 and see below for details). CR amplification through enzymatic reactions at the protein-vesicle surface We hypothesized that CR signals will further enhance after enzymatic cleavage of the protein bound at the vesicle surface. This is because the enzyme will dock with protein during cleavage process exerting additional molecular stress at the PDA membrane. The result of the molecular stress at the membrane due to vesicle enzymatic cleavage activities is expected to result in the amplification of changes in the membrane packing and conformation yielding substantial increase in the CR signal. This hypothesis was tested by adding chymotrypsin to protein-tagged-vesicles. Chymotrypsin is a serine protease which catalyzes the hydrolytic cleavage of peptide bonds under physiological conditions.49 It contains a hydrophobic pocket and an oxyanion hole that is specific to the cleaving bonds adjacent to aromatic amino



acid residues (tryptophan, phenylalanine and tyrosine).34,

49

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Indeed, we observed that a consistent

significant increase in the CR signal after addition of enzyme in the solution. The CR response-MW curve for the enzymatic-protein MW is shown in Fig. 5B. Here, CR (=CRf-CRi) denotes increase in the CR signal following enzymatic cleavage reaction at the vesicle surface. CRi and CRf are colorimetric responses before and after enzymatic cleavage of the bound protein molecules at the vesicle membrane surface. CR > 100% was observed for enzymatic cleavage of the larger MW proteins (for example, myosin and IgG) bound to vesicles. The enzymatic cleavage reaction of the smaller MW proteins (lysozyme and BSA) consistently yielded CR in 3-6% range. For example, lysozyme-bound at the vesicle membrane surface produced CR~3% following reaction with the chymotrypsin. These results also reinforce our argument that lysozyme may not be fully accessible for enzymatic reaction but is partially embedded in the membrane. The in-silico analysis (using http://web.expasy.org/peptide_mass/) suggests that there are 10, 55, and 185 possible chymotrypsin induced cleavage sites available for lysozyme, BSA and myosin.44 However, not all of these cleavage sites on protein molecules may be available for the enzymatic reactions due to steric hindrance and/or reduced accessibility of the enzyme to cleavage sites. Further, we also imagine that some enzymatic cleavage activities may not contribute significantly to the overall observed CR signal because these cleavage reactions may occur farther away from protein-membrane interface such that the stress induced through these interactions is small without producing large changes in the configuration of PDA backbone. In any case, the average number of qualitatively enzymatic cleavage events is likely to be higher for higher MW protein molecules compared to that for the smaller MW protein molecules. Overall, enzymatic cleavage of covalently bound proteins at the PDA vesicles provides an excellent opportunity to rapidly probe proteins for chromatic signaling at nanomolar concentrations. IR spectroscopic analysis 20 ACS Paragon Plus Environment

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To gain insight into microscopic arrangement of membrane packing and molecular orientation of the protein-vesicle complexes, we performed FTIR analysis on these complexes. IR spectroscopy is a useful characterization tool for probing defects in mono- and multilayer molecular systems. In general, the position, intensity, full-width-at-half-maximum (peak width), and area of CH stretching resonance vibrational bands in the alkyl chain provide important information on changes in the local molecular conformations. For example, symmetric (s,CH) and asymmetric(as,CH) C-H stretching for highly packed crystalline C15-alkanoic acids chemisorbed on metal surfaces appear at 2848 cm-1 and 2915 cm-1 respectively38, 50 whereas the corresponding gauche-conformations containing CH stretching peaks are blue-shifted to higher frequency.38 Similarly, as,CH2 and s,CH2 for lipids in bilayers and multilayers in ordered crystalline-state appear at 2917 cm-1 and 2849 cm-1, whereas those in the disordered liquidcrystalline state appear at 2923 cm-1 and 2853 cm-1 respectively (Table 3S).50 The methyl and methylene groups in the protein molecules are disordered in nature and exhibits at frequency >2918 cm-1.38 Fig. 6 and Table 3S show the as,CH and s,CH for the protein-bound and thermally treated (at 75 oC) PDA vesicles. For all protein-bound and thermally-treated PDA vesicles, both as,CH2 and s,CH2 occur at frequency 90%) of white solid. 1H NMR (300 MHz, DMSO), δ (ppm): 0.89 (t, 3H), 1.27 (m, 26H), 1.5 (m, 4H), 1.75 (m, 2H), 2.25 (t, 4H), 2.37 (m, 1H), 2.61 (m, 1H), 2.84 (s, 2H).

The synthesis of amine-

tagged sulforhodamine-101 and sulforhodamine-101 tagged PCDA were followed from the literature published.56 NHS-PDA reaction studies. The reactivity of NHS with nucleophile is histidine, imidazole > primary amino groups (either α or ε) >> cysteine thiolate = tyrosine phenolate.29 Vesicle Preparation



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Preparation of small vesicles (SV): The vesicles were synthesized according to a published literature procedure.56 We prepared four different kinds of vesicles containing various ratios of 1:1a:3:4 (Scheme 1 of main text) by first dissolving the lipid mixture in the chloroform in a round-bottom flask. The solvent was evaporated completely under reduced vacuum that yielded a thin film of lipids and monomers attached to the inner wall of the flask. Hydration of the film was accomplished using deionized water yielding a vesicle concentration of 1 mM. The suspension was sonicated at 80 °C for ∼18 min that enhanced the homogeneity of the solution by breaking up large aggregates present in the solution. The remaining aggregates in the solution were removed by passing the solution through a 0.8 μm nylon filter. The optically clear solution was kept at 4 °C overnight. The self-assembled PCDA/NHS-PCDA/DMPC monomers were irradiated using 254 nm of UV irradiation for ∼2-5 min using a Pen Ray UV source (4.5 mW/cm2) in air. The polymerized vesicles were kept in refrigerator in dark until use. Preparation of giant vesicle (GVs): Besides the use of nanoscale vesicles, we have used GVs (diameter ∼15−60 μm) for the studies involving the vesicle−BSA interactions using fluorescence microscopy. The use of GVs provided a convenient way to visually probe the interactions of vesicles with BSA. These studies clearly show that the vesicles are attached to BSA and provided useful information on BSA distribution on the surface of GVs. The self-assembled GVs were prepared by using a mixture of 1, 1a, 3, and 4 according to the following procedure.57 The mixture of lipids was dissolved in chloroform such that the total monomer concentration was 1 mM in the final solution. Optically clear solution was obtained by passing the solution through a 0.45 μm nylon filter. The dried material was then dissolved in 3 mL : 1 mL :: chloroform : methanol. A ~25 mL of nanopure water was then added carefully along the flask walls to the aqueous solution. The organic solvent was removed in a rotary evaporator under reduced pressure that yielded an opalescent fluid. The resultant solution was cooled at 4 °C overnight and kept in the refrigerator until further use.


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Optical spectroscopic measurements: The Perkin Elmer Lambda 25 UV/Vis spectrophotometer was used for absorbance analyses. The scanning speed and slit width for data acquisition was 500 nm/min and 1 nm. The emission spectra were measured using a Photon Technology International spectrometer (scanning speed 300 nm/min). For all emission spectra, the excitation wavelength (λex) of PDA and SR101 were 470 nm and 560 nm, respectively. Both the excitation and emission slit widths were 1 nm for all the experiments. Fluorescence anisotropy (FA) measurements. The purpose of anisotropic studies is to evaluate the binding reaction between vesicles and fluorophore-tagged protein molecules. Perrin equation (Eq. 1S) was used to estimate the expected FA of SR-101 before and after binding to the vesicle particles. After binding of the free fluorophores to the vesicle surface, the rotational correlation time () and FA of the fluorophore decreases drastically. FA measurements were performed for three different samples: PDA vesicles (red), PDA-NHS vesicle after SR-101 reaction, and free SR-101. Since blue PDA vesicles possess extremely low quantum yield, the red-phase PDA vesicles were more appropriate for the FA experiments. -.

=1+

-

1

Eq. 1S

2

where r0 is intrinsic anisotropy (r0 for sulfo-rhodamine = 0.37),58 r is anisotropy of SR101-tagged to vesicle  is SR-101 lifetime (4.9 ns).59 Debey-Stokes-Einstein.59 The following expression provides a relationship between rotational correlational times and viscosity. The rotational correlational time was used to obtain

calculated

anisotropy: Φ=

ɳ5

Eq. 2S

(v + h)

67

where  is the viscosity of the solvent in which fluorophore resides; M is the molecular weight of the rotating particle; R is the gas constant, and T is the solution temperature (in K). Value of  and the hydration (h) for proteins in solution are usually 0.7 ml/g and ~0.23 g H2O per gram of protein, 31 ACS Paragon Plus Environment


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respectively. For free sulfo-rhodamine 101 in buffer solution, we estimated  = 0.2 by using M = 527g/mole,  is 1.0 cP, R = 8.31x107 erg/mole K, and T = 298 K. Inserting these values in Perrins equation (Eq. 1S) gives r = 0.016 at T = 298 K. Our experimental r matches closely with the estimated r = 0.0157. Estimation of rotational correlation time of Vesicle: Following Debey-Stokes-Einstein expression, we estimated correlational rotation time  = 0.14 ms. Inserting  value to Perrins equation provided FA (r) = 0.216. This value is very similar to our experimental FA of red vesicles. Experimental values of FA for the rhodamine bonded vesicle exhibited a FA (r) = 0.10. Fluorescence resonance energy transfer (FRET) studies. Proximity check between SR-101 and vesicle. The reaction between amine–SR101 and NHS-PDA exhibits efficient FRET between SR-101 (donor) and PDA (acceptor) only if the distance between SR-101 and PDA conjugated chain is less then Forster radius (r0) for SR-101 and PDA pair. The estimated r0 between SR-101 and PDA is 2.8 nm. In general, the rate of FRET from donor to acceptor is given by the following equation:59 k>7 =

?@A BC

Eq. 3S

-D

where k is a constant, κ2 is the orientation factor, Q is the emission quantum yield of donor, J is the spectral overlap function, and r is donor-acceptor distance. The change in FRET efficiency (E) from SR-101 to PDA in the present study was due to changes in the spectral overlap (J) before and after the photopolymerization of PDA vesicle. kET was close to zero for unpolymerized liposomes and SR-101 because the resonance factor (J) for unpolymerized solution was zero. FRET efficiency (E) between SR-101 and PDA vesicle was ~72% after polymerization of vesicle, indicating close-proximity between SR-101 and PDA vesicles. Colorimetric response (CR%). CR% value is a measure of blue to red transition in the PDA vesicles after covalent-binding of proteins and enzymatic cleavage reactions. To analyze the effect of covalent 32 ACS Paragon Plus Environment

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attachment of different protein molecules on the PDA surface, the absorbance spectra of the solution before and after protein binding with vesicles was analyzed and CR values were estimated. The spectra were obtained between 450 to 750 nm at 15 ± 1°C. To quantify the extent of blue-to-red transitions following formula were used:23 BF = ! G

Eq. 4S

(

GDHI,K IH.,K L GDHI ,F

where Bi is defined as blue absorbance fraction before blue to red color transition. BM = ! G

GDHI,N IH.,N L GDHI,N

Eq. 5S

(

where Bf is defined as blue absorbance fraction after blue to red color transition. %CR = (

RK SRN

Eq. 6S

) × 100

RK

Microscopic analysis. Transmission electron micrographs (Hitachi-7100) were acquired at 60kV to visualize the vesicles and vesicle-protein complex after negative staining with uranyl acetate or phosphotungstic acid. A fluorescence microscope (Leica DMIRB) equipped with a QImaging (Cooled Mono 12-bit) CCD camera was used to visualize vesicle and vesicle-BSA complex. The size and topology of vesicles on glass surfaces were measured using an atomic force microscope (AFM) (Topometrix TMX1010). Mass spectrometry (MS) analysis. PDA-NHS vesicle solution was allowed to react with Bovine serum albumin (BSA) for 1 hour which was followed by dialysis using a membrane (MWCO 100,000 Dalton) to separate the free unbound BSA fragments from those bound to vesicles. Chymotrypsin was used to digest both the BSA-tagged vesicles and free BSA to investigate which amino acid sequence was attached to the vesicles (see below). This digested BSA-tagged vesicle solution was filtered using Millipore Ultracel-10K membrane filters to isolate the resultant peptides. A 1-L aliquot of the filtrate containing peptides was deposited with 1-L of matrix solution (5 mg/mL -cyano-4-hydroxycinnamic acid in 0.1% 33 ACS Paragon Plus Environment


Page 34 of 38

aqueous trifluoroacetic acid) on a stainless-steel sample plate and allowed to dry at room temperature. The sample plate was then introduced into a Microflex-LR time-of-flight mass spectrometer (Bruker Daltonics, Billerica, MA) and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectra were generated by firing a nitrogen laser (337 nm) in the positive ion mode. The BSA peptides were identified by comparing the observed peptide ion signal mass-to-charges against the theoretical digest of BSA by chymotrypsin using Protein Prospector online software available from the University of California–San Francisco. BSA protein structure was visualized using PyMOL molecular visualization software. vdw energy estimation: The vdW interaction energy between vesicles and protein molecules were estimated by assuming that they are hard-sphere and hard-shell.60 E  E sphere,core  E sphere,shell  E( sphere,core)  E  R1,  R2  h  ,  d  h   E( sphere,shell )  E  R2 , R1, d   E  R1, R2  h, d  h 

E

sphere,shell 

  A  123  6 

R  d 2        2  1 1         ln  A  R2  R2  2R1 2R1    123 6   R R  d2  R  d 2    d 2  2d  1 1   4R1  2d  1 1   4R1  2d  1 1   R R R R R R  2  2  2  2  2       2     h   2  1  R    R2  2   d  h    R h

 2d  1 1    R2   R2 

h

 h 2  1  R  R2  R

   h 

  4 1 R  2(d  h)  R1 1 R       R2  1  2 2 2 R  

d  h

2





R

2



 ln

 

 R1 h  d  h 1   R R2  2 R2  h  R

2

 

2d h



 4  1  R1  2(d  h)  1 1   R2    R2   R2





Rearranging above equation by noting that R2>>R1, d, h provides:


   2  d  h   R

 

2



Page 35 of 38 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


  d2 2d ( )(R R )  2 2 1 2 E  ( 123 ) ( )( ) [ln( R2 )]  2d d2 6  2d d2 vdw 2d d2 ( )  ( ) 4  ( )  ( ) 4  ( )  ( )  RR RR RR R R  R 2 1 2 2 1 2 2 1 2     2(d  h) (d  h)2  ( )  ( ) A 2 2  R2 R1R2  ( 123 ) ( )  ( ) [ln( )] 2(d  h) (d  h)2  6  2(d  h) (d  h)2 2(d  h) (d  h)2 )( RR ) 4( R )( RR ) 4( R )( R R )   ( R 2 1 2 2 1 2 2 1 2   A





Hamaker constants.

Hamaker constants for membrane-water-protein complex were estimated by the

following equation:61 A123 



A123 

 410

A11  A33 21



A22  A33

 4.41020



 1.51020  4.41020



where A11  ALipid A22  ABSA A33  AWater

Hamaker constant (A22) for all the protein was assumed to be same. It was also assumed that a thin water layer was present in-between membrane and protein. A123 value for the membrane-water-protein complex was estimated to be ~5.9x10-21 J.

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