scholarly journals A proteoliposome-based system reveals how lipids control photosynthetic light harvesting

2020 ◽  
Vol 295 (7) ◽  
pp. 1857-1866 ◽  
Author(s):  
Stefanie Tietz ◽  
Michelle Leuenberger ◽  
Ricarda Höhner ◽  
Alice H. Olson ◽  
Graham R. Fleming ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Light Harvesting ◽  
Single Photon ◽  
Pressure Profile ◽  
Cd Spectroscopy ◽  
Quantum Yields ◽  
The Impact

Integral membrane proteins are exposed to a complex and dynamic lipid environment modulated by nonbilayer lipids that can influence protein functions by lipid-protein interactions. The nonbilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in plant photosynthetic thylakoid membranes, but its impact on the functionality of energy-converting membrane protein complexes is unknown. Here, we optimized a detergent-based reconstitution protocol to develop a proteoliposome technique that incorporates the major light-harvesting complex II (LHCII) into compositionally well-defined large unilamellar lipid bilayer vesicles to study the impact of MGDG on light harvesting by LHCII. Using steady-state fluorescence spectroscopy, CD spectroscopy, and time-correlated single-photon counting, we found that both chlorophyll fluorescence quantum yields and fluorescence lifetimes clearly indicate that the presence of MGDG in lipid bilayers switches LHCII from a light-harvesting to a more energy-quenching mode that dissipates harvested light into heat. It is hypothesized that in the in vitro system developed here, MGDG controls light harvesting of LHCII by modulating the hydrostatic lateral membrane pressure profile in the lipid bilayer sensed by LHCII-bound peripheral pigments.

scholarly journals DNA-Encircled Lipid Bilayers

2018 ◽  
Author(s):  
Katarina Iric ◽  
Madhumalar Subramanian ◽  
Jana Oertel ◽  
Nayan P. Agarwal ◽  
Michael Matthies ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Protein Function ◽  
Dna Nanotechnology ◽  
Lipid Vesicles ◽  
Associated Proteins

ABSTRACTLipid bilayers and lipid-associated proteins play a crucial role in biology. As in vivo studies and manipulation are inherently difficult, several membrane-mimetic systems have been developed to enable investigation of lipidic phases, lipid-protein interactions, membrane protein function and membrane structure in vitro. Controlling the size and shape, or site-specific functionalization is, however, difficult to achieve with established membrane mimetics based on membrane scaffolding proteins, polymers or peptides. In this work, we describe a route to leverage the unique programmability of DNA nanotechnology and create DNA-encircled bilayers (DEBs), which are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle. To stabilize the hydrophobic rim of the lipid bilayer, and to prevent formation of lipid vesicles, we introduced up to 2 alkyl chains per helical that point to the inside of the toroidal DNA ring and interact with the hydrophobic side chains of the encapsulated lipid bilayer. The DEB approach described herein provides unprecedented control of size, and allows the orthogonal functionalizations and arrangement of engineered membrane nanoparticles and will become a valuable tool for biophysical investigation of lipid phases and lipid-associated proteins and complexes including structure determination of membrane proteins and pharmacological screenings of membrane proteins.

scholarly journals Isolation and structural elucidation of dimeric epigallocatechin-3-gallate autoxidation products and their antioxidant capacity

2021 ◽  
Author(s):  
Julian Alfke ◽  
Uta Kampermann ◽  
Svetlana Kalinina ◽  
Melanie Esselen
Keyword(s):  
Antioxidant Capacity ◽  
Antioxidant Properties ◽  
Cd Spectroscopy ◽  
Trolox Equivalent Antioxidant Capacity ◽  
Published Data ◽  
Dietary Polyphenols ◽  
Cellular Effects ◽  
The Impact

AbstractDietary polyphenols like epigallocatechin-3-gallate (EGCG)—which represents the most abundant flavan-3-ol in green tea—are subject of several studies regarding their bioactivity and health-related properties. On many occasions, cell culture or in vitro experiments form the basis of published data. Although the stability of these compounds is observed to be low, many reported effects are directly related to the parent compounds whereas the impact of EGCG degradation and autoxidation products is not yet understood and merely studied. EGCG autoxidation products like its dimers theasinensin A and D, “P2” and oolongtheanin are yet to be characterized in the same extent as their parental polyphenol. However, to investigate the bioactivity of autoxidation products—which would minimize the discrepancy between in vitro and in vivo data—isolation and structure elucidation techniques are urgently needed. In this study, a new protocol to acquire the dimers theasinensin A and D as well as oolongtheanin is depicted, including a variety of spectroscopic and quadrupole time-of-flight high-resolution mass spectrometric (qTOF-HRMS) data to characterize and assign these isolates. Through nuclear magnetic resonance (NMR) spectroscopy, polarimetry, and especially circular dichroism (CD) spectroscopy after enzymatic hydrolysis the complementary atropisomeric stereochemistry of the isolated theasinensins is illuminated and elucidated. Lastly, a direct comparison between the isolated EGCG autoxidation products and the monomer itself is carried out regarding their antioxidant properties featuring Trolox equivalent antioxidant capacity (TEAC) values. These findings help to characterize these products regarding their cellular effects and—which is of special interest in the flavonoid group—their redox properties.

scholarly journals Activities and regulation of peptidoglycan synthases

2015 ◽  
Vol 370 (1679) ◽  
pp. 20150031 ◽  
Author(s):  
Alexander J. F. Egan ◽  
Jacob Biboy ◽  
Inge van't Veer ◽  
Eefjan Breukink ◽  
Waldemar Vollmer
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Cytoplasmic Membrane ◽  
Current Knowledge ◽  
Protein Protein Interactions ◽  
Penicillin Binding Proteins ◽  
Multiple Protein ◽  
Cell Division Protein ◽  
The Impact

Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

scholarly journals Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

2016 ◽  
Vol 113 (46) ◽  
pp. E7185-E7193 ◽  
Author(s):  
Rahul Grover ◽  
Janine Fischer ◽  
Friedrich W. Schwarz ◽  
Wilhelm J. Walter ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Gliding Motility ◽  
Supported Lipid Bilayers ◽  
Transport Efficiency ◽  
Collective Transport ◽  
Wide Range ◽  
Motor Density

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.

scholarly journals Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7

2020 ◽  
Vol 295 (15) ◽  
pp. 5067-5080 ◽  
Author(s):  
Akello J. Agwa ◽  
Poanna Tran ◽  
Alexander Mueller ◽  
Hue N. T. Tran ◽  
Jennifer R. Deuis ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Lipid Membrane ◽  
Surface Profile ◽  
Voltage Gated Sodium Channel ◽  
Voltage Gated ◽  
Gating Modifier ◽  
Peptide Toxin

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV1.7 and with an in vivo efficacy similar to that of native HwTx-IV.

scholarly journals Hazelnut (Corylus avellana) vicilin Cor a 11: molecular characterization of a glycoprotein and its allergenic activity

2004 ◽  
Vol 383 (2) ◽  
pp. 327-334 ◽  
Author(s):  
Iris LAUER ◽  
Kay FOETISCH ◽  
Daniel KOLARICH ◽  
Barbara K. BALLMER-WEBER ◽  
Amedeo CONTI ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Corylus Avellana ◽  
Cd Spectroscopy ◽  
Mediator Release ◽  
Food Allergies ◽  
Ige Reactivity ◽  
Allergenic Activity ◽  
The Impact

In Europe, hazelnuts (Corylus avellana) are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterized. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterization of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Although MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS showed that one of two potential glycosylation sites of Cor a 11 was glycosylated, CD spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus to analyse the impact of the glycan residues of Cor a 11 on IgE binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitization pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine-release assays were used to determine the allergenic activity of both natural and recombinant Cor a 11 in comparison with Cor a 1, a birch (Betula verrucosa) pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

scholarly journals Physical mechanisms driving the reversible aggregation of Staphylococcus aureus and response to antimicrobials

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Céline Burel ◽  
Rémi Dreyfus ◽  
Laura Purevdorj-Gage
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Potential ◽  
Protein Interactions ◽  
Extracellular Proteins ◽  
Cell Protein ◽  
Reversible Aggregation ◽  
Biological Phenomena ◽  
Decreasing Ph ◽  
The Impact

AbstractFormation of non-sessile, auto-aggregated cells of Staphylococcus aureus contributes to surface colonization and biofilm formation, hence play a major role in the early establishment of infection and in tolerance to antimicrobials. Understanding the mechanism of aggregation and the impact of aggregation on the activity of antimicrobials is crucial in achieving a better control of this important pathogen. Previously linked to biological phenomena, physical interactions leading to S. aureus cellular aggregation and its protective features against antimicrobials remain unraveled. Herein, in-vitro experiments coupled with XDLVO simulations reveal that suspensions of S. aureus cells exhibit rapid, reversible aggregation (> 70%) in part controlled by the interplay between cellular hydrophobicity, surface potential and extracellular proteins. Changing pH and salt concentration in the extracellular media modulated the cellular surface potential but not the hydrophobicity which remained consistent despite these variations. A decrease in net cellular negative surface potential achieved by decreasing pH or increasing salt concentrations, caused attractive forces such as the hydrophobic and cell–protein interactions to prevail, favoring immediate aggregation. The aggregation significantly increased the tolerance of S. aureus cells to quaternary ammonium compounds (QAC). The well-dispersed cell population was completely inactivated within 30 s whereas its aggregated counterpart required more than 10 min.

scholarly journals Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

2020 ◽  
Author(s):  
Gerhard Wagner ◽  
Meng Zhang ◽  
Miao Gui ◽  
Zi-Fu Wang ◽  
Christoph Gorgulla ◽  
...  
Keyword(s):  
Complex Formation ◽  
G Protein ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Protein Complex ◽  
Lipid Membrane ◽  
Protein Complexes ◽  
Structural Basis ◽  
Downstream Signaling

Abstract G protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, structural studies of the complex in a physiological lipid membrane environment are lacking. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gai1b1g1 protein in two conformational states, resolved to 4.1 and 4.2 Å resolution. The structures were determined in lipid bilayer without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

scholarly journals Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

2016 ◽  
Author(s):  
Rahul Grover ◽  
Janine Fischer ◽  
Friedrich W. Schwarz ◽  
Wilhelm J. Walter ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Gliding Motility ◽  
Supported Lipid Bilayers ◽  
Transport Efficiency ◽  
Collective Transport ◽  
Wide Range ◽  
Biological Substrates ◽  
Motor Density

AbstractIn eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to non-biological substrates. However, the collective transport by membrane-anchored motors, i.e. motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted ‘membrane-anchored’ gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding-peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single-motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason, that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect which we directly observed using singlemolecule fluorescence microscopy. Our results illustrate the importance of the motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.

scholarly journals High efficiency excitation energy transfer in biohybrid quantum dot–bacterial reaction center nanoconjugates

2021 ◽  
Author(s):  
Giordano Amoruso ◽  
Juntai Liu ◽  
Daniel W Polak ◽  
Kavita Tiwari ◽  
Michael R Jones ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Time Scale ◽  
High Efficiency ◽  
Light Harvesting ◽  
Single Photon ◽  
Visible Region ◽  
Solar Spectrum ◽  
Direct Determination

Reaction centers (RCs) are the pivotal component of natural photosystems, converting solar energy into the potential difference between separated electrons and holes that is used to power much of biology. RCs from anoxygenic purple photosynthetic bacteria such as Rhodobacter sphaeroides only weakly absorb much of the visible region of the solar spectrum which limits their overall light-harvesting capacity. For in vitro applications such as bio-hybrid photodevices this deficiency can be addressed by effectively coupling RCs with synthetic light-harvesting materials. Here, we studied the time scale and efficiency of Förster resonance energy transfer (FRET) in a nanoconjugate assembled from a synthetic quantum dot (QD) antenna and a tailored RC engineered to be fluorescent. Time-correlated single photon counting spectroscopy of biohybrid conjugates enabled the direct determination of FRET from QDs to attached RCs on a time scale of 26.6 ± 0.1 ns and with a high efficiency of 0.75 ± 0.01.

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