A QCM-D measurements of SLB interactions with lignin dimers

Artificial membranes composed of SLBs have structures and dynamics that mimic the primary characteristics of cell membranes and are, thus, frequently used as simplified systems for studying membrane thermodynamics and transport processes.58,5958. R. P. Richter, R. Bérat, and A. R. Brisson, Langmuir 22, 3497 (2006). https://doi.org/10.1021/la052687c59. A. A. Adib, S. Nazemidashtarjandi, A. Kelly, A. Kruse, K. Cimatu, A. E. David, and A. M. Farnoud, Environ. Sci. Nano 5, 289 (2018). https://doi.org/10.1039/C7EN00685C The SALB deposition method was used as the membrane preparation approach here because it has been shown to produce reproducible bilayers on unmodified gold QCM sensors.4444. S. R. Tabaei, J.-H. Choi, G. Haw Zan, V. P. Zhdanov, and N.-J. Cho, Langmuir 30, 10363 (2014). https://doi.org/10.1021/la501534f QCM-D is a highly sensitive method, especially for viscous layers containing large amounts of water (e.g., lipid bilayers), and has been used to investigate SLB formation and molecular interactions at lipid–bilayer interfaces.5050. T. K. Lind and M. Cardenas, Biointerphases 11, 020801 (2016). https://doi.org/10.1116/1.4944830 In QCM-D, the energy dissipation is recorded as the damping of the resonance when the driving voltage to the sensor is shut off. Variations in dissipation (ΔD) are related to changes in the rigidity or viscoelasticity of the layer adhered on the surface;2424. C. M. Bailey, E. Kamaloo, K. L. Waterman, K. F. Wang, R. Nagarajan, and T. A. Camesano, Biophys. Chem. 203-204, 51 (2015). https://doi.org/10.1016/j.bpc.2015.05.006 the deformation of a soft or disordered adlayer on the substrate leads to large dissipation changes, whereas nondeformable or rigid adlayers are identified by small dissipation changes.Here, QCM-D was utilized to examine and quantify the adsorption of lignin dimers to supported lipid bilayers. Representative frequency and dissipation plots for the formation of a DPPC-supported lipid bilayer using the SALB method at 297 ± 0.2 K are illustrated in Fig. 2, where DPPC is below its gel–fluid phase transition temperature (Tm = 315.3 ± 0.15 K). In Fig. 2, the time axis starts after reaching steady state, with PBS flowing over the sensor. PBS is followed by isopropanol, followed by a solution of DPPC in isopropanol (0.5 mg/ml), and finally PBS again. Rinsing with PBS in the final step causes the lipids adsorbed to the sensor from the isopropanol solution to reorganize into a bilayer,4444. S. R. Tabaei, J.-H. Choi, G. Haw Zan, V. P. Zhdanov, and N.-J. Cho, Langmuir 30, 10363 (2014). https://doi.org/10.1021/la501534f as shown by a sharp increase in frequency and a corresponding sharp decrease in dissipation. The Δf value for the bilayer in Fig. 2 before dividing by the overtone number (n = 3) is ∼ −62 Hz, which, using the Sauerbrey equation [Eq. (1)], corresponds to a mass value of ∼366 ng/cm2, falling within the mass range reported in the literature for a supported lipid bilayer.60,6160. C. A. Keller and B. Kasemo, Biophys. J. 75, 1397 (1998). https://doi.org/10.1016/S0006-3495(98)74057-361. R. Richter, A. Mukhopadhyay, and A. Brisson, Biophys. J. 85, 3035 (2003). https://doi.org/10.1016/S0006-3495(03)74722-5 The ΔD for the gel-phase DPPC bilayer in Fig. 2(b) is 10.6 × 10−6, which is higher than the value reported for fluid-phase lipid bilayers (less than 0.5 × 10−6);4444. S. R. Tabaei, J.-H. Choi, G. Haw Zan, V. P. Zhdanov, and N.-J. Cho, Langmuir 30, 10363 (2014). https://doi.org/10.1021/la501534f however, it is consistent with the value reported by Lind et al. for gel-phase DPPC lipid bilayers in PBS at 298 K (∼6.5 × 10−6 at n = 7).6262. T. K. Lind, M. Cárdenas, and H. P. Wacklin, Langmuir 30, 7259 (2014). https://doi.org/10.1021/la500897x The mass of the SLB formed on the sensor was measured from the difference in the frequency of the initial PBS baseline (t = 5 min) and the baseline in PBS after SLB formation (t = 150 min) using the Sauerbrey equation [Eq. (1)]. The DPPC lipid bilayer mass for all SLBs in this work was above 350 ng/cm2, which is in agreement with the reported mass for a rigid SLB that is completely covered and is well coupled to the sensor.47,63,6447. K. Evans, Int. J. Mol. Sci. 9, 498 (2008). https://doi.org/10.3390/ijms904049863. T. J. Zwang, W. R. Fletcher, T. J. Lane, and M. S. Johal, Langmuir 26, 4598 (2010). https://doi.org/10.1021/la100275v64. J. T. Marquês, A. S. Viana, and R. F. M. de Almeida, Langmuir 30, 12627 (2014). https://doi.org/10.1021/la503086aThe amount of the lignin dimers interacting with the bilayer, and consequently the ratio of the mass of the bound dimer to the mass of the lipid bilayer, depends on the concentration of the dimer in solution. Because QCM sensors are sensitive to the density and viscosity of fluids, it is conventional to flow dilute solutions (with concentrations in the order of 1–1000 μM) over the sensor when studying the adsorption of molecules to lipid bilayers.21,34,6521. T. Joshi, Z. X. Voo, B. Graham, L. Spiccia, and L. L. Martin, Biochim. Biophys. Acta Biomembr. 1848, 385 (2015). https://doi.org/10.1016/j.bbamem.2014.10.01934. K. Kannisto, L. Murtomäki, and T. Viitala, Colloids Surf. B 86, 298 (2011). https://doi.org/10.1016/j.colsurfb.2011.04.01265. H.-H. Shen, P. G. Hartley, M. James, A. Nelson, H. Defendi, and K. M. McLean, Soft Matter 7, 8041 (2011). https://doi.org/10.1039/c1sm05287j Concentration-dependent studies of G-βO4′-truncG were conducted (Fig. 3) to understand the sensitivity of QCM to the contributions of uptake by the SLB as well as bulk solvent properties. QCM-D measurements were carried out using three different concentrations of G-βO4′-truncG solutions in isopropanol/PBS (0.67% v/v) in increasing order (0.01, 0.02, and 0.1 mg/ml). In Fig. 3, t = 0 corresponds to the initial-supported DPPC bilayer and the values of Δf/3 and ΔD are shifted to 0 at this initial condition. At t = 12 min, the flow of the 0.01 mg/ml dimer solution is initiated over the SLB for 1 h, resulting in a decrease in frequency (Fig. 3), which is indicative of dimer association and incorporation into the lipid bilayer. However, the frequency does not plateau over the 1 h period of injection, meaning that equilibrium with the SLB is not reached. Similarly, increasing the dimer solution concentration to 0.02 mg/ml for 1 h results in a further decrease in frequency without any evidence of membrane saturation. When the flow is switched to a much more concentrated solution of dimer (0.1 mg/ml), the frequency continues to decrease and then plateaus at ∼268 min. In the final step, the sensor was rinsed with PBS solution for ∼115 min. Thus, the concentration of 0.1 mg/ml was chosen to study the interaction of the dimers with the SLB within a time period of 1 h, with a goal of achieving measurable equilibriumlike interactions of the bilayer in semidilute solutions.To investigate the effect of the modifications of the lignin GG dimer chemical structures on the interactions with phospholipid membranes, 0.1 mg/ml solutions of benzG-βO4′-G, G-βO4′-truncG, and G-βO4′-G in isopropanol/PBS (0.67% v/v) were separately introduced to the supported DPPC bilayers on QCM sensors. The resulting QCM-D responses for Δf/3 and ΔD as functions of time are presented in Fig. 4, in which the time of zero has been set for the fully developed bilayer and a corresponding Δf/3 value of zero is set. As shown in Fig. 4(a), upon the introduction of the benzG-βO4′-G dimer to the bilayer at ∼7 min, Δf/3 drops rapidly due to a change in the solvent properties of the semidilute isopropanol-dope dimer solution (0.1 mg/ml). Concurrent with the decrease in frequency, a step change followed by a more gradual increase occurs in the dissipation profile [Fig. 4(b)]. Within 1 h of initiating the benzG-βO4′-G dimer flow over the bilayer, Δf/3 continues to slowly decrease due to the uptake of the dimer by the bilayer (causing an increase in film mass). A subsequent switch to pure PBS solution at ∼67 min results in a rapid increase in the frequency. This is mainly attributed to a difference in the bulk solvent properties and a possible redissolution of some of the dimers. Concurrently, ΔD undergoes a rapid drop immediately after switching to pure PBS and then gradually plateaus at a relatively constant value above the starting value, suggesting that the final SLB is more fluid than the initial bilayer and retains some amount of dimer.The net frequency change in the bilayer due to the binding and incorporation of the lignin dimers was interpreted from the change in frequency representing the initial DPPC-supported bilayer (before ∼7 min) and the bilayer after rinsing the sensor with pure PBS (at ∼130 min). benzG-βO4′-G and G-βO4′-truncG displayed similar trends. The net Δf/3 changes for the benzG-βO4′-G and G-βO4′-truncG were ∼ −10.13 and ∼ −3.17 Hz, respectively. Similarly, the net ΔD changes of the bilayer for the benzG-βO4′-G and G-βO4′-truncG dimers were ∼3.4 × 10−6 and ∼11.5 × 10−6, respectively [Fig. 4(b)]. The dissipation shifts measured by QCM-D are an indirect measurement of bilayer fluidity and its viscous losses.6161. R. Richter, A. Mukhopadhyay, and A. Brisson, Biophys. J. 85, 3035 (2003). https://doi.org/10.1016/S0006-3495(03)74722-5 The increase of dissipation with the introduction of benzG-βO4′-G and G-βO4′-truncG is consistent with dimer incorporation into the bilayer and suggests that these hydrophobic dimers ultimately perturb the bilayer's rigidity and correspond to an increase in dissipation loss to the surrounding medium.6666. E. Tellechea, D. Johannsmann, N. F. Steinmetz, R. P. Richter, and I. Reviakine, Langmuir 25, 5177 (2009). https://doi.org/10.1021/la803912p The results of a separate control experiment presented in Figs. S1(a) and S1(b)6969. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001029 for 1H NMR and mass spectrometry (HR-MS) of the G-βO4′-G and benzG-βO4′-G dimers structures, QCM-D results for the interaction of isopropanol with supported DPPC lipid bilayers, solvent-assisted formation of DOPC bilayers and G-bO4′-truncG dimer interaction with DOPC lipid bilayer. Supplementary material for molecular dynamics includes error estimation for partial density profiles for lignin and lipid groups as a function of dimer distance from the lipid bilayer center, bilayer thickness with high concentrations of G-bO4′-truncG dimer, top view of DPPC lipid bilayer with G-bO4′-G dimer, deuterium order parameters for carbons in the lipid tails for G-bO4′-G, benzG-bO4′-G, and G-bO4′-truncG systems, and PMF profile as a function of the distance (dL-B) from the bilayer center to the terminal –OH group of a benzG-βO4′-G. show that the use of a small amount of isopropanol to solubilize the dimers (0.67 vol. % in the PBS solution) has a minimal effect on the net frequency (−0.66 Hz) and net dissipation (less than 0.5 × 10−6) relative to the dimer uptake in the SLBs.At a concentration of 0.1 mg/ml, the interaction of G-βO4′-G with SLBs is different from that of benzG-βO4′-G and G-βO4′-truncG, as interpreted from their respective frequency and dissipation responses (Fig. 4). As seen in Fig. 4(a), G-βO4′-G incorporates more slowly into the membrane. Interestingly, Δf/3 increases when switching to the PBS buffer but does not plateau over the 1-h period of buffer rinse. Instead, it trends toward the initial frequency. The reduced changes in net frequency for the G-βO4′-G dimer (∼ −1.94 Hz) relative to benzG-βO4′-G and G-βO4′-truncG and the ability to resolubilize it during rinsing suggest that G-βO4′-G did not associate appreciably with the bilayer interior. The small net changes in ΔD (less than 1 × 10−6) indicate that the bilayer fluidity of the SLB was largely unaffected by the G-βO4′-G dimer addition and subsequent rinsing with PBS. Thus, the time-dependent desorption behavior suggests that the G-βO4′-G dimer is not well incorporated into the SLB relative to the G-βO4′-G dimer, G-βO4′-truncG.Table I summarizes the mass of dimers associated with the bilayers after the final PBS rinse relative to the mass of the supported bilayer initially deposited onto the sensor. The results are normalized with respect to bilayer mass because the bilayer mass varied slightly with each bilayer formed using the SALB method (an average of 400 ± 96 ng/cm2), which is consistent with a range reported in the literature for the mass of the supported phosphatidylcholine lipid bilayers60,6160. C. A. Keller and B. Kasemo, Biophys. J. 75, 1397 (1998). https://doi.org/10.1016/S0006-3495(98)74057-361. R. Richter, A. Mukhopadhyay, and A. Brisson, Biophys. J. 85, 3035 (2003). https://doi.org/10.1016/S0006-3495(03)74722-5 and with the observation that DPPC-supported lipid bilayers deposited in the gel phase have more variability than those deposited in the fluid phase.6262. T. K. Lind, M. Cárdenas, and H. P. Wacklin, Langmuir 30, 7259 (2014). https://doi.org/10.1021/la500897x The ratio of adsorbed dimer mass to DPPC bilayer mass follows the following trend: G-βO4′-G βO4′-truncG benzG-βO4′-G. This is consistent with the order of increasing equilibrium partitioning of these dilute lignin compounds into phospholipid bilayers6767. E. Boija and G. Johansson, Biochim. Biophys. Acta Biomembr. 1758, 620 (2006). https://doi.org/10.1016/j.bbamem.2006.04.007 as well as their estimated hydrophobicity. Our previous differential scanning calorimetry and MD studies of dimer incorporation into dilute systems also showed enhanced partitioning of the benzylated and truncated GG dimers into DPPC relative to a naturally occurring G-βO4′-G dimer.3232. X. Tong, M. Moradipour, B. Novak, P. Kamali, S. O. Asare, B. L. Knutson, S. E. Rankin, B. C. Lynn, and D. Moldovan, J. Phys. Chem. B 123, 8247 (2019). https://doi.org/10.1021/acs.jpcb.9b05525 G-βO4′-G has the most hydroxyl groups (four), one of which is a part of the hydroxypropenyl (HOC3H4−) tail. The free hydroxyl groups increase the hydrophilicity of the dimer and consequently decrease its penetration depth into the interior of the lipid bilayer, leading to slow diffusion away from the bilayer following the PBS rinse. In a study by Tammela et al., flavonoids with more than three free hydroxyl groups (e.g., luteolin, quercetin, and morin) were reported to initially bind strongly to the heads of the DPPC lipid bilayer via hydrogen bonding, but not to diffuse and transport deeply into the hydrophobic interior of the bilayers compared with more hydrophobic flavonoids.6868. P. Tammela, L. Laitinen, A. Galkin, T. Wennberg, R. Heczko, H. Vuorela, J. P. Slotte, and P. Vuorela, Arch. Biochem. Biophys. 425, 193 (2004). https://doi.org/10.1016/j.abb.2004.03.023 Here, G-βO4′-truncG lacks the hydroxypropenyl tail, which increases its hydrophobicity and ultimately leads to more penetration. Similarly, benzG-βO4′-G has an added benzyl group at the phenolic end of the dimer, which causes an increase in hydrophobicity, allowing it to penetrate deeper into the bilayer. Our previously published PMF calculations for dilute dimeric systems in fluid-phase DPPC bilayers illustrated that a major part of the G-βO4′-G dimer resides at or close to the exterior bilayer surface with only a small probability of finding this dimer inside the bilayer.3232. X. Tong, M. Moradipour, B. Novak, P. Kamali, S. O. Asare, B. L. Knutson, S. E. Rankin, B. C. Lynn, and D. Moldovan, J. Phys. Chem. B 123, 8247 (2019). https://doi.org/10.1021/acs.jpcb.9b05525 In contrast, a major part of G-βO4′-truncG is embedded in the bilayer with only a small probability of finding it at the exterior bilayer surfaces. Our PMF studies also showed that benzG-βO4′-G is expected to reside almost exclusively in the bilayer interior.3232. X. Tong, M. Moradipour, B. Novak, P. Kamali, S. O. Asare, B. L. Knutson, S. E. Rankin, B. C. Lynn, and D. Moldovan, J. Phys. Chem. B 123, 8247 (2019). https://doi.org/10.1021/acs.jpcb.9b05525

TABLE I. Quantitative QCM-D results of the lignin GG dimers' interactions with the synthetic DPPC lipid bilayers with 1 h of exposure at a concentration of 0.1 mg/ml of dimer solutions.

DimerG-βO4′-GaG-βO4′-truncGbbenzG-βO4′-GbAverage ratio of the mass of the bound dimer to the mass of the pure DPPC bilayer0.10 ± 0.0090.16 ± 0.030.25 ± 0.08The MD simulation studies reported here were performed at T = 326 K to allow for equilibration within the time scale of the simulations. However, the QCM-D experiments were performed at 297 K, which is below Tm of DPPC (Tm = 315.3 ± 0.15 K), because preliminary experiments indicated that SALB formation of a DPPC bilayer was unsuccessful at 318 K, a temperature above its Tm (data not given). To show that our QCM results were also valid for fluid bilayers, the interaction of G-βO4′-truncG as the model compound with the phosphatidylcholine lipid bilayer, DOPC (Tm = 256.65 K) was investigated at 297 K. Representative QCM-D results for solvent-assisted DOPC lipid bilayer formation on a gold sensor and the resulting frequency and dissipation profiles after the introduction of G-βO4′-truncG to the bilayer are included in Figs. S2 and S3 (Ref. 6969. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001029 for 1H NMR and mass spectrometry (HR-MS) of the G-βO4′-G and benzG-βO4′-G dimers structures, QCM-D results for the interaction of isopropanol with supported DPPC lipid bilayers, solvent-assisted formation of DOPC bilayers and G-bO4′-truncG dimer interaction with DOPC lipid bilayer. Supplementary material for molecular dynamics includes error estimation for partial density profiles for lignin and lipid groups as a function of dimer distance from the lipid bilayer center, bilayer thickness with high concentrations of G-bO4′-truncG dimer, top view of DPPC lipid bilayer with G-bO4′-G dimer, deuterium order parameters for carbons in the lipid tails for G-bO4′-G, benzG-bO4′-G, and G-bO4′-truncG systems, and PMF profile as a function of the distance (dL-B) from the bilayer center to the terminal –OH group of a benzG-βO4′-G.), respectively. The frequency and dissipation values for a DOPC lipid bilayer formed on gold were found to be −15 ± 1.5 Hz and (0.94 ± 0.2) × 10−6, respectively [Figs. S2(a) and S2(b)]6969. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001029 for 1H NMR and mass spectrometry (HR-MS) of the G-βO4′-G and benzG-βO4′-G dimers structures, QCM-D results for the interaction of isopropanol with supported DPPC lipid bilayers, solvent-assisted formation of DOPC bilayers and G-bO4′-truncG dimer interaction with DOPC lipid bilayer. Supplementary material for molecular dynamics includes error estimation for partial density profiles for lignin and lipid groups as a function of dimer distance from the lipid bilayer center, bilayer thickness with high concentrations of G-bO4′-truncG dimer, top view of DPPC lipid bilayer with G-bO4′-G dimer, deuterium order parameters for carbons in the lipid tails for G-bO4′-G, benzG-bO4′-G, and G-bO4′-truncG systems, and PMF profile as a function of the distance (dL-B) from the bilayer center to the terminal –OH group of a benzG-βO4′-G.. The net change in frequency due to the incorporation of G-βO4′-truncG into the DOPC lipid bilayer in the fluid phase and the average ratio of the mass of the adsorbed G-βO4′-truncG dimer to the mass of the DOPC bilayer were ∼ −2.33 Hz [Fig. S3(a)]6969. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001029 for 1H NMR and mass spectrometry (HR-MS) of the G-βO4′-G and benzG-βO4′-G dimers structures, QCM-D results for the interaction of isopropanol with supported DPPC lipid bilayers, solvent-assisted formation of DOPC bilayers and G-bO4′-truncG dimer interaction with DOPC lipid bilayer. Supplementary material for molecular dynamics includes error estimation for partial density profiles for lignin and lipid groups as a function of dimer distance from the lipid bilayer center, bilayer thickness with high concentrations of G-bO4′-truncG dimer, top view of DPPC lipid bilayer with G-bO4′-G dimer, deuterium order parameters for carbons in the lipid tails for G-bO4′-G, benzG-bO4′-G, and G-bO4′-truncG systems, and PMF profile as a function of the distance (dL-B) from the bilayer center to the terminal –OH group of a benzG-βO4′-G. and 0.20 ± 0.09 (Table S16969. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001029 for 1H NMR and mass spectrometry (HR-MS) of the G-βO4′-G and benzG-βO4′-G dimers structures, QCM-D results for the interaction of isopropanol with supported DPPC lipid bilayers, solvent-assisted formation of DOPC bilayers and G-bO4′-truncG dimer interaction with DOPC lipid bilayer. Supplementary material for molecular dynamics includes error estimation for partial density profiles for lignin and lipid groups as a function of dimer distance from the lipid bilayer center, bilayer thickness with high concentrations of G-bO4′-truncG dimer, top view of DPPC lipid bilayer with G-bO4′-G dimer, deuterium order parameters for carbons in the lipid tails for G-bO4′-G, benzG-bO4′-G, and G-bO4′-truncG systems, and PMF profile as a function of the distance (dL-B) from the bilayer center to the terminal –OH group of a benzG-βO4′-G.), respectively. These values are consistent with our measurements of G-βO4′-truncG dimer interactions with DPPC bilayers in the gel phase. This suggests that the phase of the lipid bilayer (gel or fluid) does not drastically affect the amount of dimers associated with the membrane. However, the net ΔD changes for the G-βO4′-truncG dimer and the DOPC bilayer were ∼1 × 10−6 [Fig. S3(b)]6969. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001029 for 1H NMR and mass spectrometry (HR-MS) of the G-βO4′-G and benzG-βO4′-G dimers structures, QCM-D results for the interaction of isopropanol with supported DPPC lipid bilayers, solvent-assisted formation of DOPC bilayers and G-bO4′-truncG dimer interaction with DOPC lipid bilayer. Supplementary material for molecular dynamics includes error estimation for partial density profiles for lignin and lipid groups as a function of dimer distance from the lipid bilayer center, bilayer thickness with high concentrations of G-bO4′-truncG dimer, top view of DPPC lipid bilayer with G-bO4′-G dimer, deuterium order parameters for carbons in the lipid tails for G-bO4′-G, benzG-bO4′-G, and G-bO4′-truncG systems, and PMF profile as a function of the distance (dL-B) from the bilayer center to the terminal –OH group of a benzG-βO4′-G., which is substantially lower than what was measured for the G-βO4′-truncG dimer and DPPC. The uptake of the G-βO4′-truncG dimer has a minimal effect on the fluidity and viscoelastic properties of a bilayer that is already in the fluid phase.