Alternatives to Styrene- and Diisobutylene-Based Copolymers for Membrane Protein Solubilization via Nanodisc Formation.

Styrene-maleic acid copolymers (SMAs), and related amphiphilic copolymers, are promising tools for isolating and studying integral membrane proteins in a native-like state. However, they do not exhibit this ability universally, as several reports have found that SMAs and related amphiphilic copolymers show little to no efficiency when extracting specific membrane proteins. Recently, it was discovered that esterified SMAs could enhance the selective extraction of trimeric Photosystem I from the thylakoid membranes of thermophilic cyanobacteria; however, these polymers are susceptible to saponification that can result from harsh preparation or storage conditions. To address this concern, we herein describe the development of α-olefin-maleic acid copolymers (αMAs) that can extract trimeric PSI from cyanobacterial membranes with the highest extraction efficiencies observed when using any amphiphilic copolymers, including diisobutylene-co-maleic acid (DIBMA) and functionalized SMA samples. Furthermore, we will show that αMAs facilitate the formation of photosystem I-containing nanodiscs that retain an annulus of native lipids and a native-like activity. We also highlight how αMAs provide an agile, tailorable synthetic platform that enables fine-tuning hydrophobicity, controllable molar mass, and consistent monomer incorporation while overcoming shortcomings of prior amphiphilic copolymers.

Effects of Esterified Styrene-Maleic Acid Copolymer Degradation on Integral Membrane Protein Extraction.

The detergent-free extraction of integral membrane proteins using styrene-maleic acid copolymers (SMAs) has shown promise as a potentially effective technique to isolate proteins in a more native-like conformation. As the field continues to develop, the protein selectivity and extraction efficiency of many analogues of traditional SMAs are being investigated. Recently, we discovered that the monoesterification of SMAs with alkoxy ethoxylate sidechains drastically affects the bioactivity of these copolymers in the extraction of photosystem I from the cyanobacterium Thermosynechococcus elongatus. However, subsequent investigations also revealed that the conditions under which these esterified SMA polymer analogues are prepared, purified, and stored can alter the structure of the alkoxy ethoxylate-functionalized SMA and perturb the protein extraction process. Herein, we demonstrate that the basic conditions required to solubilize SMA analogues may lead to deleterious saponification side reactions, cleaving the sidechains of an esterified SMA and dramatically decreasing its efficacy for protein extraction. We found that this process is highly dependent on temperature, with polymer samples being prepared and stored at lower temperatures exhibiting significantly fewer saponification side reactions. Furthermore, the effects of small-molecule impurities and exposure to light were also investigated, both of which are shown to have significant effects on the polymer structure and/or protein extraction process.

PEDOT-Carbon Nanotube Counter Electrodes and Bipyridine Cobalt (II/III) Mediators as Universally Compatible Components in Bio-Sensitized Solar Cells Using Photosystem I and Bacteriorhodopsin.

In nature, solar energy is captured by different types of light harvesting protein-pigment complexes. Two of these photoactivatable proteins are bacteriorhodopsin (bR), which utilizes a retinal moiety to function as a proton pump, and photosystem I (PSI), which uses a chlorophyll antenna to catalyze unidirectional electron transfer. Both PSI and bR are well characterized biochemically and have been integrated into solar photovoltaic (PV) devices built from sustainable materials. Both PSI and bR are some of the best performing photosensitizers in the bio-sensitized PV field, yet relatively little attention has been devoted to the development of more sustainable, biocompatible alternative counter electrodes and electrolytes for bio-sensitized solar cells. Careful selection of the electrolyte and counter electrode components is critical to designing bio-sensitized solar cells with more sustainable materials and improved device performance. This work explores the use of poly (3,4-ethylenedioxythiophene) (PEDOT) modified with multi-walled carbon nanotubes (PEDOT/CNT) as counter electrodes and aqueous-soluble bipyridine cobaltII/III complexes as direct redox mediators for both PSI and bR devices. We report a unique counter electrode and redox mediator system that can perform remarkably well for both bio-photosensitizers that have independently evolved over millions of years. The compatibility of disparate proteins with common mediators and counter electrodes may further the improvement of bio-sensitized PV design in a way that is more universally biocompatible for device outputs and longevity.

Protein Extraction Efficiency and Selectivity of Esterified Styrene-Maleic Acid Copolymers in Thylakoid Membranes.

Amphiphilic styrene-maleic acid copolymers (SMAs) have been shown to effectively extract membrane proteins surrounded by an annulus of native membrane lipids via the formation of nanodiscs. Recent reports have shown that 2-butoxyethanol-functionalized SMA derivatives promote the extraction of membrane proteins from thylakoid membranes, whereas unfunctionalized SMA is essentially ineffective. However, it is unknown how the extent of functionalization and identity of sidechains impact protein solubilization and specificity. Herein, we show that the monoesterification of an SMA polymer with hydrophobic alkoxy ethoxylate sidechains leads to an increased solubilization efficiency (SE) of trimeric photosystem I (PSI) from the membranes of cyanobacterium Thermosynechococcus elongatus. The specific SMA polymer used in this study, PRO 10235, cannot encapsulate single PSI trimers from this cyanobacterium; however, as it is functionalized with alkoxy ethoxylates of increasing alkoxy chain length, a clear increase in the trimeric PSI SE is observed. Furthermore, an exponential increase in the SE is observed when >50% of the maleic acid repeat units are monoesterified with long alkoxy ethoxylates, suggesting that the PSI extraction mechanism is highly dependent on both the number and length of the attached side chains.

Characterization and evolution of tetrameric photosystem I from the thermophilic cyanobacterium Chroococcidiopsis sp TS-821.

Photosystem I (PSI) is a reaction center associated with oxygenic photosynthesis. Unlike the monomeric reaction centers in green and purple bacteria, PSI forms trimeric complexes in most cyanobacteria with a 3-fold rotational symmetry that is primarily stabilized via adjacent PsaL subunits; however, in plants/algae, PSI is monomeric. In this study, we discovered a tetrameric form of PSI in the thermophilic cyanobacterium Chroococcidiopsis sp TS-821 (TS-821). In TS-821, PSI forms tetrameric and dimeric species. We investigated these species by Blue Native PAGE, Suc density gradient centrifugation, 77K fluorescence, circular dichroism, and single-particle analysis. Transmission electron microscopy analysis of native membranes confirms the presence of the tetrameric PSI structure prior to detergent solubilization. To investigate why TS-821 forms tetramers instead of trimers, we cloned and analyzed its psaL gene. Interestingly, this gene product contains a short insert between the second and third predicted transmembrane helices. Phylogenetic analysis based on PsaL protein sequences shows that TS-821 is closely related to heterocyst-forming cyanobacteria, some of which also have a tetrameric form of PSI. These results are discussed in light of chloroplast evolution, and we propose that PSI evolved stepwise from a trimeric form to tetrameric oligomer en route to becoming monomeric in plants/algae.

Photoelectrochemistry of photosystem I bound in nafion.

Developing a solid state Photosystem I (PSI) modified electrode is attractive for photoelectrochemical applications because of the quantum yield of PSI, which approaches unity in the visible spectrum. Electrodes are constructed using a Nafion film to encapsulate PSI as well as the hole-scavenging redox mediator Os(bpy)2Cl2. The photoactive electrodes generate photocurrents of 4 µA/cm(2) when illuminated with 1.4 mW/cm(2) of 676 nm band-pass filtered light. Methyl viologen (MV(2+)) is present in the electrolyte to scavenge photoelectrons from PSI in the Nafion film and transport charges to the counter electrode. Because MV(2+) is positively charged in both reduced and oxidized states, it is able to diffuse through the cation permeable channels of Nafion. Photocurrent is produced when the working electrode is set to voltages negative of the Os(3+)/Os(2+) redox potential. Charge transfer through the Nafion film and photohole scavenging at the PSI luminal surface by Os(bpy)2Cl2 depends on the reduction of Os redox centers to Os(2+) via hole scavenging from PSI. The optimal film densities of Nafion (10 µg/cm(2) Nafion) and PSI (100 µg/cm(2) PSI) are determined to provide the highest photocurrents. These optimal film densities force films to be thin to allow the majority of PSI to have productive electrical contact with the backing electrode.

Poly(styrene-co-maleic acid)-mediated isolation of supramolecular membrane protein complexes from plant thylakoids.

Derivatives of poly(styrene-co-maleic acid) (pSMA), have recently emerged as effective reagents for extracting membrane protein complexes from biological membranes. Despite recent progress in using SMAs to study artificial and bacterial membranes, very few reports have addressed their use in studying the highly abundant and well characterized thylakoid membranes. Recently, we tested the ability of twelve commercially available SMA copolymers with different physicochemical properties to extract membrane protein complexes (MPCs) from spinach thylakoid membrane. Based on the efficacy of both protein and chlorophyll extraction, we have found five highly efficient SMA copolymers: SMA® 1440, XIRAN® 25010, XIRAN® 30010, SMA® 17352, and SMA® PRO 10235, that show promise in extracting MPCs from chloroplast thylakoids. To further advance the application of these polymers for studying biomembrane organization, we have examined the composition of thylakoid supramolecular protein complexes extracted by the five SMA polymers mentioned above. Two commonly studied plants, spinach (Spinacia oleracea) and pea (Pisum sativum), were used for extraction as model biomembranes. We found that the pSMAs differentially extract protein complexes from spinach and pea thylakoids. Based on their differential activity, which correlates with the polymer chemical structure, pSMAs can be divided into two groups: unfunctionalized polymers and ester derivatives.

Self-assembling peptide detergents stabilize isolated photosystem I on a dry surface for an extended time.

We used a class of designed peptide detergents to stabilize photosystem I (PS-I) upon extended drying under N2 on a gold-coated-Ni-NTA glass surface. PS-I is a chlorophyll-containing membrane protein complex that is the primary reducer of ferredoxin and the electron acceptor of plastocyanin. We isolated the complex from the thylakoids of spinach chloroplasts using a chemical detergent. The chlorophyll molecules associated with the PS-I complex provide an intrinsic steady-state emission spectrum between 650 and 800 nm at -196.15 degrees C that reflects the organization of the pigment-protein interactions. In the absence of detergents, a large blue shift of the fluorescence maxima from approximately 735 nm to approximately 685 nm indicates a disruption in light-harvesting subunit organization, thus revealing chlorophyll-protein interactions. The commonly used membrane protein-stabilizing detergents, N-dodecyl-beta-D-maltoside and N-octyl-beta-D-glucoside, only partially stabilized the approximately 735-nm complex with approximately 685-nm spectroscopic shift. However, prior to drying, addition of the peptide detergent acetyl-AAAAAAK at increasing concentration significantly stabilized the PS-I complex. Moreover, in the presence of acetyl-AAAAAAK, the PS-I complex is stable in a dried form at room temperature for at least 3 wk. Another peptide detergent, acetyl-VVVVVVD, also stabilized the complex but to a lesser extent. These observations suggest that the peptide detergents may effectively stabilize membrane proteins in the solid-state. These designed peptide detergents may facilitate the study of diverse types of membrane proteins.

Ultrasonic spectroscopy and differential scanning calorimetry of liposomal-encapsulated nisin.

The thermal stability of phosphatidylcholine (PC) liposomes (colloidal dispersions of bilayer-forming polar lipids in aqueous solvents) in the presence and absence of the antimicrobial polypeptide nisin was evaluated using differential scanning calorimetry (DSC) and low-intensity ultrasonic spectroscopy (US). PC liposome mixtures with varying acyl chain lengths (C16:0 and C18:0) were formed in buffer with or without entrapped nisin. Gel-to-liquid crystalline phase transition temperatures (T(M)) of liposomes determined from DSC thermograms were in excellent agreement with those determined by ultrasonic velocity and attenuation coefficient measurements recorded at 5 MHz. The dipalmitoylphosphatidylcholine (DPPC) T(M) measured by DSC was approximately 41.3 and approximately 40.7 degrees C when measured by ultrasonic spectroscopy. The T(M) of distearoylphosphatidylcholine (DSPC) and DPPC/DSPC 1:1 liposomes was 54.3 and 54.9 degrees C and approximately 44.8 and approximately 47.3 degrees C when measured by DSC and US, respectively. The thermotropic stability generally increased upon addition of nisin. Analysis of the stepwise decrease in ultrasonic velocity with temperature indicated an increased compressibility corresponding to a loss of structure upon heating.

Size, stability, and entrapment efficiency of phospholipid nanocapsules containing polypeptide antimicrobials.

The effect of lipid composition [phosphatidylcholine (PC), phosphatidylglycerol (PG), and cholesterol] on size, stability, and entrapment efficiency of polypeptide antimicrobials in liposomal nanocapsules was investigated. PC, PC/cholesterol (70:30), and PC/PG/cholesterol (50:20:30) liposomes had entrapment efficiencies with calcein of 71, 57, and 54% with particle sizes of 85, 133, and 145 nm, respectively. Co-encapsulation of calcein and nisin resulted in entrapment efficiencies of 63, 54, and 59% with particle sizes of 144, 223, and 167 nm for PC, PC/cholesterol (70:30), and PC/PG/cholesterol (50:20:30), respectively. Co-encapsulation of calcein and lysozyme yielded entrapment efficiencies of 61, 60, and 61% with particle sizes of 161, 162, and 174 nm, respectively. The highest concentration of antimicrobials was encapsulated in 100% PC liposomes. Nisin induced more calcein release compared to lysozyme. Results demonstrate that production and optimization of stable nanoparticulate aqueous dispersions of polypeptide antimicrobials for microbiological stabilization of food products depend on selection of suitable lipid-antimicrobial combinations.

Comparative photoactivity and stability of isolated cyanobacterial monomeric and trimeric Photosystem I.

Photoactivity of native trimeric, and non-native monomeric Photosystem I (PSI) extracted from Thermosynechococcus elongatus is compared in a photoelectrochemical system. The PSI monomer is isolated by disassembling a purified PSI trimer using a freeze-thaw technique in presence of the short-chain surfactant, octylthioglucoside. Photoactive electrodes are constructed with PSI, functioning as both light absorber and charge-separator, embedded within a conductive polymer film. Despite structural differences between PSI trimers and monomers, electrodes cast with equal chlorophyll-a concentration generate similar photoactivities. Photoaction spectra show that all photocurrent derived from electrodes of PSI and conductive polymer originates solely from PSI with no photocurrent caused by redox mediators in the conductive polymer film. Longevity studies show that the two forms of PSI photodegrade at the same rate while exposed to equal intensities of 676 nm light. Direct photo-oxidation measurements indicate that PSI's monomeric form has fewer light harvesting antennae than trimer, and also shows energy sharing between monomeric subunits in the trimer. The structure of PSI is also found to impact cell performance when applying light at incident powers above 1.5 mW/cm(2) due to the reduced optical cross-section in the monomer, causing saturation at lower light intensities than the trimer.

Liposomal nanocapsules in food science and agriculture.

Liposomes, spherical bilayer vesicles from dispersion of polar lipids in aqueous solvents, have been widely studied for their ability to act as drug delivery vehicles by shielding reactive or sensitive compounds prior to release. Liposome entrapment has been shown to stabilize encapsulated, bioactive materials against a range of environmental and chemical changes, including enzymatic and chemical modification, as well as buffering against extreme pH, temperature, and ionic strength changes. Liposomes have been especially useful to researchers in studies of various physiological processes as models of biological membranes in both eukaryotes and prokaryotes. Industrial applications include encapsulation of pharmaceuticals and therapeutics, cosmetics, anti-cancer and gene therapy drugs. In the food industry, liposomes have been used to deliver food flavors and nutrients and more recently have been investigated for their ability to incorporate food antimicrobials that could aid in the protection of food products against growth of spoilage and pathogenic microorganisms. In this review we briefly introduce key physicochemical properties of liposomes and review competing methods for liposome production. A survey of non-agricultural and food applications of liposomes are given. Finally, a detailed up-to-date summary of the emerging usage of liposomes in the food industry as delivery vehicles of nutrients, nutraceuticals, food additives, and food antimicrobials is provided.