Revealing the Nature of Interaction between Graphene Oxide and Lipid Membrane by Surface-Enhanced Infrared Absorption Spectroscopy

Revealing the nature of interaction between graphene oxide (GO) and lipid membrane is a crucial issue but still remains challenging. Here, we describe our recent effort toward this direction by studying the GO-induced vibrational changes of interfacial water and lipid membrane with surface-enhanced infrared absorption (SEIRA) spectroscopy. The experimental results provide evidence that overcoming the electrostatic repulsion of phosphate group, its hydrogen bonding attraction as well as the electrostatic and hydrophobic interaction of choline group are the driving forces for the effective adsorption of GO on lipid membrane. This work will open exciting new avenues to explore the use of SEIRA spectroscopy technique in probing nanobio interface

The structure of FClip1 and sequence alignment of FClip1 with related α/β hydrolases in the NC-loop region.

<p>(A) Secondary structure topology diagram of FClip1. The α-helices and β-sheets are represented by cylinders and arrows, respectively. The α/β hydrolase fold domain is shown in gray and white, the cap domain is shown in blue, and the NC-loop is shown in red. The locations of the conserved residues of the catalytic machinery are shown in orange. (B) The modeled structure of FClip1. The catalytic triad Ser107, His267, and Asp239 are shown in ball and sticks. (C) Sequence alignment of FClip1 with other lipases and perhydrolases in the NC-loop region. The residues of other lipases similar to FClip1 are indicated by the inverted triangles. The residues of the perhydrolases similar to FClip1 are indicated by triangles. The highly conserved residues are indicated by stars. Q9KJG6: lipase lip3 from <i>Pseudomonas aeruginosa</i> LST-03; Q9AMF7: triacylglycerol acyl hydrolase from <i>Moritella marina</i>; P24640: lipase lip3 from <i>Moraxella</i> sp. TA144; Q02104: triacylglycerol lipase from <i>Psychrobacter immobilis</i>; Q35A75: probable lipase from <i>Bradyrhizobium</i> sp. BTAi1; Q93MV2: lipase from <i>Streptococcuss</i> sp. N1; A1YV97: lipase FClip1 from <i>Fervidobacterium changbaicum</i>; O31168: non-haem chloroperoxidase from <i>Streptomyces aureofaciens</i>; P49323: chloroperoxidase from <i>Streptomyces lividans</i>; P33912: bromoperoxidase BPO-A1 from <i>Streptomyces aureofaciens</i>; O31158: non-heme chloroperoxidase from <i>Pseudomonas fluorescens</i>; P29715: bromoperoxidase BPO-A2 from <i>Streptomyces aureofaciens</i>.</p

Thermodynamic parameters of the wild type FClip1 and the NC-loop deletion mutants.

a<p>Numbers in brackets indicate the values relative to wild type.</p

Understanding the Synergic Mechanism of Weak Interactions between Graphene Oxide and Lipid Membrane Leading to the Extraction of Lipids

Revealing how weak forces interact synergistically to induce differences in nanobio effects is critical to understanding the nature of the nanobio interface. Herein, graphene oxide (GO) and a lipid membrane are selected as a nanobio model, and interaction forces at the GO–biomembrane interface are modulated by varying the amounts and species of oxygenated functional groups on the surface of GO. A synergic mechanism of interfacial interaction forces is investigated by a combination of surface-enhanced infrared absorption (SEIRA) spectroscopy, confocal laser scanning microscopy (CLSM), and electrochemical impedance spectroscopy (EIS). The results reveal that after balancing with electrostatic repulsion, the moderate attraction between GO and lipid headgroups (such as electrostatic and/or hydrophobic interactions) is most favorable for lipid extraction, whereas lipid extraction is inhibited under an attraction that is too strong or too weak. Under moderate attraction between GO and the headgroups of lipids, the appropriate degree of rotation freedom is maintained for GO, which is beneficial to the hydrogen-bonding interaction between the CO group in the phosphatide hydrophobic region and GO, thus triggering the insertion of GO into the lipid alkyl chain region, resulting in the rapid and significant extraction of lipids. Our results have important guiding significance for how to reveal the synergistic mechanism of weak interactions at the nanobio interface

Role of the NC-Loop in Catalytic Activity and Stability in Lipase from <em>Fervidobacterium changbaicum</em>

<div><p>Flexible NC-loops between the catalytic domain and the cap domain of the α/β hydrolase fold enzymes show remarkable diversity in length, sequence, and configuration. Recent investigations have suggested that the NC-loop might be involved in catalysis and substrate recognition in many enzymes from the α/β hydrolase fold superfamily. To foster a deep understanding of its role in catalysis, stability, and divergent evolution, we here systemically investigated the function of the NC-loop (residues 131–151) in a lipase (FClip1) from thermophilic bacterium <em>Fervidobacterium changbaicum</em> by loop deletion, alanine-scanning mutagenesis and site-directed mutagenesis. We found that the upper part of the NC-loop (residues 131–138) was of great importance to enzyme catalysis. Single substitutions in this region could fine-tune the activity of FClip1 as much as 41-fold, and any deletions from this region rendered the enzyme completely inactive. The lower part of the NC-loop (residues 139–151) was capable of enduring extensive deletions without loss of activity. The shortened mutants in this region were found to show both improved activity and increased stability simultaneously. We therefore speculated that the NC-loop, especially the lower part, would be a perfect target for enzyme engineering to optimize the enzymatic properties, and might present a hot zone for the divergent evolution of α/β hydrolases. Our findings may provide an opportunity for better understanding of the mechanism of divergent evolution in the α/β hydrolase fold superfamily, and may also guide the design of novel biocatalysts for industrial applications.</p> </div

Specific activities and kinetic parameters of the wild type FClip1 and the deleted mutants.

<p>Specific activities were measured in 50 mM phosphate buffer (pH 8.0) at 75°C using <i>p</i>NPC4 and <i>p</i>NPC12 as the substrates, respectively. Kinetic parameters were obtained in 50 mM phosphate buffer (pH 8.0) at 75°C using <i>p</i>NPC4 as the substrate. The fitting curves for kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046881#pone.0046881.s006" target="_blank">Figure S6</a>.</p>a<p>Numbers in brackets indicate the values relative to wild type.</p

Specific activities and kinetic parameters of the wild type FClip1 and its mutants.

<p>Enzyme assays were performed in 50 mM phosphate buffer (pH 8.0) at 75°C using <i>p</i>NPC4 as the substrate. The fitting curves for kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046881#pone.0046881.s006" target="_blank">Figure S6</a>.</p>a<p>Numbers in brackets indicate the values relative to wild type.</p

Design of the NC-loop deletion mutagenesis of FClip1.

<p>(A) Detailed structural conformation of the NC-loop. The upper part of the NC-loop (Asp131–Ser138), which is buried inside the protein, is shown in yellow; the lower part (Glu139–Lys151), which is exposed in the solvent, is shown in red. (B) The design of systematic deletion of the NC-loop.</p