ArginineâPhosphate Recognition Enhanced in Phospholipid

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C: Surfaces, Interfaces, Porous Materials, and Catalysis

Arginine-Phosphate Recognition Enhanced in Phospholipid Monolayers at Aqueous Interfaces Jennifer F. Neal, Wei Zhao, Alexander J. Grooms, Amar H Flood, and Heather Cecile Allen J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b03531 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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The Journal of Physical Chemistry

Arginine-Phosphate Recognition Enhanced in Phospholipid Monolayers at Aqueous Interfaces Jennifer F. Neal,† Wei Zhao,‡ Alexander J. Grooms,† Amar H. Flood,*‡ Heather C. Allen*† †

Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210,

United States ‡

Department of Chemistry, Indiana University, Bloomington, IN 47405, United States

1 ACS Paragon Plus Environment

The Journal of Physical 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: Due to the growing world population, there is an ever-increasing need to develop better receptors to recover and recycle phosphate for use in agricultural processes. This need is driven by agricultural demand and environmental concerns because phosphate eutrophication has a damaging effect on fresh water supplies by fueling algal blooms. The air/water interface provides a unique region with a dielectric constant (ε) that diminishes from high in bulk water (ε = 80) to significantly lower (e.g., ε < 40) near the monolayer surface to potentially enhance affinities during molecular recognition. The work presented here uses a model system of phosphate binding to an amino acid, arginine, and utilizes the interfacial properties of the phospholipid monolayer, 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), as the phosphate source to quantify binding. Employing arginine as a probe molecule allows for the evaluation of its guanidinium moiety for phosphate chelation. Surface pressure–area isotherms from Langmuir monolayer studies, and corresponding infrared reflection absorption spectroscopy (IRRAS), were used along with Brewster angle microscopy (BAM) for in situ determination of molecular binding interactions and the surface binding constants of the phosphate-guanidinium complex, which are shown here to be greater than 103 M–1. The binding constant in bulk solution, determined by Nuclear Magnetic Resonance (NMR) titrations of phosphate and arginine, is determined to be on the order of 0.1 M–1. The greater than 10,000fold increase from bulk aqueous solution to the air/water interface reveals that the interface provides a region of enhanced binding affinity.


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INTRODUCTION Phosphorus, used as an agricultural fertilizer, is an essential nutrient for crops.1 But excess phosphorus in the form of phosphate runoff has detrimental effects on water quality1–4 and represents a net loss to the phosphate cycle. To prevent these negative impacts, there is a continued need to remove phosphate from natural waters by understanding its recognition.5–12 For this purpose, the lipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA, Figure 1a) and amino acid arginine (Figure 1b) were chosen with an interest in understanding the binding (Figure 1c) of phosphates using guest molecules with both environmental,13,14 sustainability,2 and biological15–17 outcomes. In addition, this type of interfacial recognition has the potential for use in sensing,18,19 molecular devices,20,21 and biology-based applications.22–24 The interactions between phosphate and amino acids such as arginine also have great significance for the understanding of molecular transfer mechanisms of marine aerosols derived from the sea surface, where lipids and amino acids are abundant.25–31 The DPPA molecule forms an insoluble monolayer, allowing for the systematic evaluation of phosphate binding mechanisms in a confined environment: the air/water interface. Arginine, a soluble, charged amino acid possessing guanidinium, ammonium, and carboxylate moieties, is an informative guest molecule. Both molecules, DPPA and arginine, possess vibrational handles that are also accessible by surface spectroscopy. Arginine’s guanidinium moiety allows a hydrogen bonding network with six possible interactions for binding,15 and the pKa of the guanidinium moiety in water is ~13.6,32 which renders it positively charged in unpolluted, natural waters (pH ~6.5-8.5)33 to support both hydrogen bonding and electrostatic interactions with a potential phosphate host.34 However, it has been shown that electrostatic interactions are screened by the solvent dielectric constant,35 which in turn leads to less effective binding



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interactions in aqueous systems as a result of strong hydration.36 To counteract these penalties to binding, there is a definite change in the hydration environment when going from bulk water to the air/water interface,37 but they have rarely been studied. The study of recognition at aqueous interfaces continues to be an open area of investigation. At interfaces, electrical double layers emerge, multivalency38 can have an impact, the structure of water is altered,39–43 and new driving forces can emerge to enrich or deplete solutes at the interface relative to the bulk.44–50 Studies addressing the impact of these factors on recognition, however, are relatively few.38,51–56 The correlation of those nominally two-dimensional affinities to the ones for related equilibria in bulk solution is a feature that needs to be addressed for a deeper understanding of recognition.36 Nevertheless, we wish to examine and confirm the remarkable and poorly understood phenomenon of enhanced affinity of the charged guest solute for the interface prior to establishing correlations between bulk and surface binding. The air/water interface is a region with a lower dielectric constant than bulk water, which is expected to cause an enhancement in the electrostatics of binding.37 Model systems of phosphate derivatives interacting with guanidinium have been considered.57–63 Previous work by Kunitake showed there is a strengthening in binding affinity between phosphate and a guanidinium host depending on whether this interaction occurs in bulk water, at the surface of micelles, or at the monolayer surface.37,58,64–66 The reduced dielectric constants that are believed to emerge at monolayer-water interfaces to help enhance binding have been addressed with a two-medium model.67–69 The interfacial dielectric constant has been estimated experimentally using a polaritysensitive fluorescent dye incorporated into a vesicle composed of phosphatidylcholine and was found to be ε ~35 at pH 6.38,53 Ratiometric probes have also shown interfacial dielectric constants


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to vary by cell type with mitochondria, being ~7.7 in one cell line and ~30 in another.70 Other membranes examined include the endoplasmic reticulum at ε ~20.71 To determine the binding constant at the air/water interface, prior work has employed ex situ methods by transferring Langmuir-Blodgett films of phosphate bound to guanidinium monolayers and taking X-ray photoelectron spectra of these films.37,58

It is possible that

transferring the monolayer disrupts the true nature of the binding event.72 With this issue in mind, the methods presented here are in situ to preserve the integrity of the aqueous interfacial binding interaction to provide the needed understanding of recognition chemistry.36 Thus, we test Kunitake’s hypothesis of enhanced affinity at interfaces by in situ methods, as well as the generality of the idea by using a system of inverted charge, that is, a negatively charged monolayer instead of positively charged. The work presented here offers new insights into the phosphate-guanidinium interaction of a simple phospholipid monolayer binding to arginine. Binding constants were quantified in situ using surface pressure area isotherms and infrared reflection absorption spectroscopy (IRRAS) that respectively provide dynamic and static measurements. We systematically studied arginine, guanidinium, and glycine to evaluate the binding interactions of these structurally different guest moieties (Figure 1b). The interface is seen to enhance binding by over four orders of magnitude relative to bulk aqueous solution when forming the putative charge-assisted hydrogen bonds (Figure 1d). This finding supports hypotheses presented from ex situ studies58 that interfaces enhance affinity.

METHODS



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Materials. All materials were commercially available and used without further purification. A stock solution of 1,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt (DPPA) (≥99%, Sigma) was dissolved in a mixture of 4:1 chloroform:methanol (v/v) (HPLC grade, Fisher Scientific). LArginine monohydrochloride (≥99.5%, Sigma), glycine (≥99.0%, Sigma), and guanidine hydrochloride (≥99%, Sigma) were dissolved in fresh ultrapure water with a resistivity of 18.2 MΩ·cm (Barnstead Nanopure Filtration System, model D4741, Barnstead-Thermolyne Corporation, Dubuque, IA). The measured pH for the highest concentration of each solution tested is pH 6.4 for arginine, pH 6.4 for glycine, and pH 6.5 for guanidinium, and these pH values are the maximum deviations from pure water. At near neutral pH, the ammonium (amino group on glycine pKa is 9.6)73 and guanidinium moieties are positivity charged, although the authors acknowledge that the protonation state at the interface may be somewhat different than bulk solution.29 Ethylenediaminetetraacetic acid (EDTA) (99.995%, Sigma) was also used in control studies to eliminate trace metals (Figure S1). Sodium dihydrogen phosphate (monohydrated ≥99.5%, Sigma) and deuterium oxide (D2O, 99.96%, Cambridge Isotopes) were purchased for Nuclear Magnetic Resonance (NMR) experiments. Deuterium oxide (D2O, 99%, Sigma) was purchased for IRRAS experiments. Surface Pressure-Area Isotherms. Surface pressure-area (Π-A) isotherms were performed on a custom Teflon Langmuir trough with an area of 144.5 cm2 equipped with movable Delrin barriers (KSV NIMA, Finland). Surface pressure was measured by the Wilhelmy plate method using filter paper plates (Ashless grade, Whatman), and controlled using KSV software (KSV NIMA, Finland). The DPPA monolayer was deposited onto the surface dropwise using a microsyringe (Hamilton, United States). To allow for solvent evaporation, 10 minutes elapsed before the start of every experiment. The compression speed was kept at 5 mm/min per barrier.


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For experiments done at constant pressure, once the desired surface pressure was reached, the barriers moved slowly at a rate of 1 mm/min per barrier both forward and backward to maintain the surface pressure. The concentration of the initial solution of DPPA on water was calibrated to liftoff at 47.5 Å2/molecule according to previous work done in the Allen group.74 All experiments were run at 21.8 °C ± 0.7 °C and a relative humidity of 31% ± 7%. Brewster Angle Microscopy. Brewster angle microscopy (BAM) and Π -A isotherms were performed concurrently using a custom-built Brewster angle microscope.75,76 The 5 mW He−Ne laser source (Research Electro-Optics, Boulder, CO) emits polarized light at 543 nm with linear polarization. The beam goes through a half-waveplate (Ekspla, Lithuania) and a Glan polarizer in which the combination of the two optics allow attenuation with polarization and purification of the p-polarized light before being reflected off the aqueous surface. The Brewster angle microscope is mounted on a goniometer to adjust the incident angle to the Brewster angle of ~53° for an aqueous surface and was adjusted slightly for amino acid solutions. The reflected beam goes through a 10X infinity-corrected super-long working distance objective lens (CFI60 TU Plan EPI, Nikon Instruments, Melville, NY) and a tube lens (MXA22018, Nikon Instruments; focal length 200 mm) to collect and collimate the beam before going into a backilluminated EM-CCD camera (iXon DV887-BV, Andor Technology USA, Concord, MA; 512 × 512 active pixels with 16 µm × 16 µm pixel size).

The dark regions of the images

correspond to the aqueous surface or gaseous region of the lipid while the bright regions of the images correspond to the lipid domains. The BAM images shown here were processed using ImageJ software77 and cropped from their original size to show the region of highest resolution. Infrared Reflection–Absorption Spectroscopy. Infrared reflection–absorption spectroscopy (IRRAS) spectra were recorded using an FT-IR spectrometer (Spectrum 100, Perkin Elmer,



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United States) with a liquid nitrogen cooled HgCdTe (MCT) detector. The Langmuir trough was placed inside the spectrometer on a breadboard with two gold plated mirrors that were adjusted to collect the reflectivity off the monolayer at an incident angle of 46o. Spectra were plotted as reflectance–absorbance (RA) which is given as

RA = –log(Rm/Ro) where Rm is the reflectivity

of the monolayer and Ro is the reflectivity of the subphase solution surface. Each spectrum was recorded as an average of 300 scans collected using unpolarized light in single beam mode. The data processing was done using Origin (OriginLab 9, Northampton, MA). Since the phosphate region has a slanted baseline, each spectrum was background subtracted by fitting a line across points 1215 cm–1 to 1134 cm–1. The spectra shown here are the result of averaging at least three spectra using the average function in Origin. For spectra obtained using D2O to avoid atmospheric H2O exchange and minimize H2O bending mode interferences, petri dishes were used instead of the trough and the sample compartment was thoroughly purged with dry nitrogen gas before the experiments. 1

H Nuclear Magnetic Resonance (NMR) Titration. All the 1H nuclear magnetic resonance

(NMR) spectra were recorded on a 600 MHz Varian Inova NMR spectrometer. A solution of arginine amino acid (100 mM, D2O) was prepared in a silicon septum sealed NMR tube and an initial spectrum was taken. A solution of sodium dihydrogen phosphate (NaH2PO4, 2.5 M, D2O) was also prepared and added to the solution of arginine amino acid with known quantities, the spectrum was recorded after each addition. All the spectroscopic data were analyzed by using MestReNova software.

RESULTS AND DISCUSSION


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Arginine-DPPA Binding Interactions at the Air/Water Interface. The goal of this work is to present new quantitative methods to study the binding affinity and molecular interactions of amphiphilic phospholipid molecules binding to a guanidinium guest, arginine, at the aqueous interface. Surface pressure–mean molecular area isotherms (Π-A) and IRRAS provides a means to study the binding properties in situ at the aqueous interface. As shown below, we determine here that the binding affinity of the phospholipid to arginine is greater than 103 M–1 at the interface, which is significantly larger than that determined for bulk solution of phosphate and arginine. Π-A isotherms of the DPPA monolayer spread onto bulk solutions of increasing concentrations of arginine (0.001 – 20 mM, Figure 2, left panel) show a gradual expansion to larger apparent mean molecular areas (MMA) from 45 to 50 Å2/molecule. The shape of the isotherm in the condensed phase for the DPPA monolayer also changes with increasing addition of arginine. The isotherm of DPPA on pure water shows distinct phases with decreasing MMA (accessed from right to left on the isotherm curve): the gaseous (G)-liquid condensed (LC) coexistence, tilted liquid condensed (TC), un-tilted liquid condensed (UC), and collapse (Figure S2). The highly-condensed UC phase that is observed in the isotherm of DPPA on pure water is either shifted to higher MMAs or absent completely at high concentrations of arginine. Results from the expansion of the monolayer and phase transition changes in the Π-A isotherms both strongly suggest there is a binding interaction occurring between DPPA and arginine. To differentiate between binding of the guanidinium on the side chain of arginine and the ammonium moiety of the amino group, Π-A isotherms with control compound glycine in the subphase were tested at concentrations up to 100 mM (Figure 2, right panel). Although there is not a significant change in the isotherms observed in these glycine control experiments, there is a



small expansion of approximately 0.43 Å2 (taken at 10 mN/m) at 100 mM glycine.

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This

expansion could arise from ammonium binding to the phosphate headgroup on account of the fact that the ammonium also provides a hydrogen bonding donor to a potential phosphate host. Previous studies showed that synthetic receptors with an ammonium functionality are effective hosts for phosphate binding in bulk aqueous solution, so it is expected that the ammonium on the amino acid binds to the phospholipid molecules to some extent.78,79 However, the results from the control experiment with glycine suggests that the guanidinium functional group is mainly responsible for the expansion observed in the isotherms. The expansion is also observed with 5 mM guanidinium in the subphase (Figure S3), which further supports this hypothesis. To further explore the binding interactions spectroscopically we employed IRRAS (Figure 3). IRRAS spectra of the phosphate region reveal the asymmetric PO2– stretch (νas PO2–) of the DPPA molecule at 1167 cm–1.80,81

The phosphate stretching mode has been shown to be

sensitive to hydration82–85 and binding,84 and is therefore a potentially selective probe of the DPPA-arginine binding interactions. The νas PO2– mode of DPPA was interrogated as a function of increasing arginine, glycine, and guanidinium concentrations. Glycine and guanidinium were chosen to differentiate between the multiple binding moieties of arginine that could interact with DPPA. Three independently collected spectra were averaged for each concentration in these plots. It is observed that the phosphate peak of DPPA increases in both intensity and broadness upon arginine addition which strongly suggests that arginine is binding to the phosphate (– RHPO4–) headgroup of DPPA. A similar result is observed with the addition of guanidinium, suggesting that the arginine interaction results from the guanidinium moiety (Figure 3, bottom left panel). There are minimal changes in the phosphate peak intensity observed with glycine addition, with the exception of 100 mM (Figure 3, top right panel). At that high concentration,


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the peak broadness and intensity increased which suggests the ammonium could also act as a binding unit for DPPA, albeit at a concentration 10,000 times that of the comparable spectral change of arginine. In order to quantify the binding observed from these results, affinities were determined based on both the peak intensity of the asymmetric stretch from the IRRAS spectra, and the mean molecular expansion (MME) of DPPA at 10 mN/m from the Π-A isotherms (Figure S4). The error bars on these plots correspond to one standard deviation, and this standard deviation was propagated through addition or division in the calculation and shown as error on the normalized plots. Results were fit to a generalized Langmuir model, with an assumption that the binding occurred as a 1:1 ratio of DPPA to arginine:86–88 [Arginine]

= K + [Arginine] (eq 1) where I and are the intensity and the maximum intensity, respectively, of the νas PO2– mode of DPPA or the MME of DPPA at 10 mN/m after subtraction from its respective intensity on water, [Arginine] is the bulk concentration of arginine, and K is the apparent equilibrium dissociation constant. The normalized plots with propagated error and simulated binding curves are shown in Figure 4 and the apparent binding affinities are summarized in Table 1. Both surface methods, Π-A isotherms and IRRAS, show a binding affinity greater than 103 M–1. There are several factors that could influence the magnitude of this apparent binding constant, in which the normalization process for the model is critical in the calculation. The data was fit to the highest intensity instead of normalizing the intensity of the highest concentration tested. Based on this, “0” corresponds to the intensity of DPPA on water, and “1” corresponds to the highest intensity recorded. The method followed here lowers the magnitude of the apparent binding constant, and 11 ACS Paragon Plus Environment


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provides more confidence in the fitting. Consequently, the binding affinity represents the lower end because of the assumptions made in the fitting. Nevertheless, the binding affinity of 103 M–1 is still very large. These results demonstrate that the hydration penalties that hinder binding in bulk aqueous solution are greatly reduced at the interface. Binding Affinity of Phosphate and Arginine in Bulk Water. The binding affinity between sodium dihydrogen phosphate (NaH2PO4) and arginine in bulk solution was investigated using nuclear magnetic resonance (NMR) titrations in deuterium oxide (Figure 5). The crystal structure of arginine and phosphate in the solid state89 shows phosphate interacts with arginine by cooperation from multiple hydrogen bonding interactions between phosphate and both the guanidinium and ammonium moieties. Upon addition of sodium dihydrogen phosphate into concentrated solution of arginine (100 mM, 298 K), the chemical shift of protons (Ha, Hd) on arginine are shifted slightly upfield (Figure 5). These results are indicative of weak interaction between arginine and phosphate that result from the highly competitive hydration of both arginine and phosphate. Chemical shifts were tracked as a function of equivalents of sodium dihydrogen phosphate added to the solution. Even with a large amount of phosphate, the change in chemical shift of arginine is still not saturated, which corresponds to weak binding to phosphate. Binding was too weak to determine using standard techniques and was therefore estimated from simulations to be 0.1 M–1 in both unbuffered and buffered (HEPES) aqueous solutions (see SI, Figures S5-S12). NMR titration in bulk solution shows weak binding between arginine and phosphate; however, as seen in results from Π-A isotherms and IRRAS shown above, the binding affinity is highly improved at air/water interfaces (Table 1). Arginine-DPPA Binding Mechanism Probed by IRRAS and BAM. The mechanistic properties of the binding interactions in the DPPA and arginine system were studied. BAM


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images in the collapse phase help to clarify the monolayer loss mechanisms (e.g. homogeneity versus 3D aggregation and/or crystallization). Figure 6 shows the BAM images of the DPPA in the collapse region of the isotherm at the air/water interface. The collapse phase region of the isotherm (Figure S2) is defined as the region where the monolayer film undergoes a 2D to 3D transition; it is the point beyond full compression of the DPPA monolayer such that no additional compression can be exerted without loss of material from the monolayer, either into 3D aggregates or loss into the bulk.90 The morphology of DPPA in the condensed region leading up to the collapse phase does not show a difference with or without arginine in the aqueous subphase (Figure S13). In Figure 6, the top left BAM image is that of DPPA spread on a pure water subphase revealing a homogenous nondescript monolayer. However, upon evaluation of the DPPA spread on aqueous solutions with arginine, the collapse phase showed the appearance of a striated crystalline morphology for low arginine concentrations (top right 2 images of Figure 6).

For the higher

arginine concentrations tested, the striated collapsed structures were minimal and the appearance of a homogenous surface is observed, similar to that which we observe from the DPPA on the pure water surface. The difference in morphology of the DPPA monolayer indicates that the binding motif in the collapse phase depends on the concentration of arginine. As observed in the Π-A isotherms and the IRRAS, there is a saturation of arginine binding that corresponds well with the change in morphology observed in these BAM images. In order to further elucidate the binding motif between arginine and DPPA and to probe the carbonyl region of DPPA, it was necessary to perform IRRAS experiments using D2O to prevent the bending mode of water (H2O) from obscuring this spectral region.91,92 The carbonyl band is very sensitive to hydration. Spectra were taken in petri dishes at a MMA of approximately 46



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Å2/molecule in the TC region of DPPA. With DPPA spread on the purely D2O subphase, plotting of the carbonyl region shows the unhydrated carbonyl band at 1738 cm–1 and the hydrated carbonyl peak at 1720 cm–1.93,94 A decrease in the hydrated carbonyl stretch is observed for DPPA on 10 mM arginine in D2O, which suggests a change in the carbonyl environment (Figure 7). We expect that since the guanidinium moiety of arginine is interacting with the phosphate headgroup of DPPA as discussed above (Figure 3) it is highly plausible that the ammonium moiety is now in close proximity with the DPPA monolayer and free to bind with one of the two carbonyls in DPPA. This binding interaction could change the carbonyl environment of DPPA and lead to the decrease in hydration observed in the IRRAS spectra. There are additional peaks observed in the IRRAS spectra of DPPA with arginine at approximately 1605 cm–1 and 1585 cm–1. These peaks are assigned to the ν (C–N) modes of arginine’s guanidinium moiety, and there is also likely overlap with the ν (COO–) mode of the amino acid.95–97 The peaks are shifted from their typical frequency in bulk water as a result of deuterium exchange occurring between the labile protons on arginine and D2O.96 As a consequence of the nature of the IRRAS experiment, spectra were recorded with arginine in the solution as a background and used as the initial reflectivity in the reflectance-absorbance equation. With spectra plotted as reflectance-absorbance and arginine in the initial solution, the presence of these arginine-based peaks in the spectra supports the hypothesis that arginine approaches the surface and binds to the DPPA molecules. The result of these additional IRRAS experiments further support the hypothesis that arginine binds to the molecules of DPPA at the interface through multiple hydrogen bonding interactions. Binding Enhancement at the Interface. The higher binding observed at interfaces versus bulk is consistent with separate reports by Kunitake,37 by Hunter,38 and others.53 We attribute


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this observation, based on their work and the reported lower dielectric,38,53,70,71 to the lower dielectric constant at the DPPA interface. The corresponding free energy of arginine association to the monolayer of –17 kJ mol–1 represents a reaction equilibrium. While it reflects the strength of the binding site composed of charge-assisted hydrogen bonding units (e.g., the phosphate head of DPPA and the guanidinium moiety of the arginine) it also reflects any structural rearrangements in the molecular monolayer, at the level of the molecule and the superstructure, as well as of any solvation changes associated with production of the complex from the solvated interface and individually solvated guests in bulk solution. The proposed binding modes (Figure 1c) involve charge-assisted hydrogen bonding and can provide some perspective on the interaction energies. The reaction free energy can be compared to similar recognition motifs in similar environments. It is on the same order of magnitude seen by others, including the binding of phosphate in bulk solution to guanidiniumbased receptors. Schmidtchen has reported98 binding of phosphate (H2PO4–) to a receptor with a single guanidinium motif on order –24 kJ mol–1 in acetonitrile (ε ~ 37). The polarity of the medium and the strength of the single binding site are on a similar order of magnitude to the monolayer system studied herein. However, deeper correlations between the apparent binding energies at the interface to the actual binding interactions on the molecular level would require further study, such as may be provided by use of a homologous series of receptors varying only in one feature. Other topics for future analysis will be investigations aimed at conversion of the apparent equilibrium dissociation constant, K, to a two-dimensional equilibrium constant and to the intrinsic binding strength at the interface.38,53,99,100

CONCLUSIONS 15 ACS Paragon Plus Environment


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Aqueous interfaces composed of a monolayer of the model phospholipid DPPA shows significantly enhanced phosphate-arginine binding relative to bulk aqueous solution. This finding is supported by in situ surface sensitive techniques including Π-A isotherms and IRRAS that were used here to quantitatively measure the apparent binding affinity of DPPA to an arginine guest. Binding affinities are found to be significantly higher (103 M–1) than that determined for bulk solution (0.1 M–1). This large binding affinity difference between surface and bulk strongly suggests that the aqueous interface enhances binding for systems with large hydration penalties. This finding is consistent with the diminished dielectric constants seen at similar interfaces. Arginine, glycine, and guanidinium were systematically chosen as guest molecules to evaluate the binding of the phosphate moiety of DPPA in monolayers to the ammonium and guanidinium functional groups of arginine. Results from IRRAS clearly show that the guanidinium functional group of arginine is primarily responsible for the large binding observed in this system. This study also demonstrates the utility of Π-A isotherms, IRRAS, and BAM as effective techniques for the evaluation of binding at the surface of aqueous solutions for systems that might have low affinities in bulk solution. To the author’s knowledge this is the first IRRAS study that quantifies binding affinity.

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Π-A isotherm and IRRAS control studies using EDTA, Π-A isotherm with guanidinium chloride, 1H NMR titrations of arginine and phosphate with and without HEPES buffer, simulated bulk binding plots using Hyss, pH titrations of arginine with sodium salts (NaH2PO4,


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NaCl and NaClO4), 1H NMR titrations of arginine and sodium salts (NaH2PO4, NaCl and NaClO4). AUTHOR INFORMATION Corresponding authors *E-mail: [email protected]. Phone: +1-812-856-3642. Fax: +1-812-855-8300. *Email: [email protected]. Phone: +1-614-292-4707. Fax: +1-614-292-1685. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This research was supported under grant CHE 1609672 from the National Science Foundation.

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FIGURES

Figure 1. Structures of the materials used in this study: (a) 1,2-dipalmitoyl-sn-glycero-3phosphatidic acid (DPPA), (b) L-arginine monohydrochloride, guanidine hydrochloride, and 30 ACS Paragon Plus Environment

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glycine. Proposed binding modes of arginine with (c) DPPA at air/water interfaces, and (d) sodium phosphate in bulk water.

Figure 2. Surface pressure compression isotherms of DPPA monolayers on different concentrations of arginine (left panel) and glycine (right panel). The isotherm on water for both plots is shown for comparison.



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Figure 3. IRRAS spectra of the phosphate peak (νas PO2–) of DPPA showing the intensity increase as a function of arginine (top left panel) and guanidinium concentrations (bottom left panel). A minimal effect is seen with increasing glycine concentrations up to 101 mM (top right panel).


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Figure 4. Normalized mean molecular area expansion of DPPA monolayers on different concentrations of arginine relative to water in the TC phase (10 mN/m) reveal an increase with arginine addition until saturation of binding sites (left). The normalized νas PO2– intensity of DPPA at 1167 cm–1 reveals an increase in intensity with arginine addition until saturation of binding sites (right).



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Figure 5. 1H NMR titrations of arginine (100 mM, 600 MHz, 298 K) with sodium dihydrogen phosphate in D2O. The proton chemical shift is shown to decrease depending on the increasing amount of dihydrogen phosphate, indicating very weak binding in bulk water.


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Figure 6. BAM images of DPPA in the collapse phase. Top panel from left to right: DPPA spread on pure water, on arginine (0.01 mM), and on arginine (0.1 mM). Bottom panel from left to right: DPPA spread on higher concentrations of arginine (1 mM), arginine (5 mM), and arginine (10 mM). The scale bar is 100 microns.



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Figure 7. IRRAS spectra of DPPA in the TC phase on D2O and arginine in D2O. Carbonyl peaks show dehydration upon arginine addition, and presence of guanidinium peaks from arginine further suggests binding interactions between DPPA and arginine.

Table 1. Summary of the Binding Affinities of Phosphate to Arginine Using Π-A Isotherms and IRRAS as in situ Interfacial Techniques, and NMR for Bulk Solution Method

Binding affinity (M–1)

Π-A isotherms (surface)

2.3 (0.9) × 103

IRRAS (surface)

6.8 (2.8) × 103

1

H NMR (bulk)

0.1


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ArginineâPhosphate Recognition Enhanced in Phospholipid

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ArginineâPhosphate Recognition Enhanced in Phospholipid