Modulation of physiological and pathological activities of lysozyme by biological membranes

AbstractThe molecular details of interactions between lipid membranes and lysozyme (Lz), a small polycationic protein with a wide range of biological activities, have long been the focus of numerous studies. The biological consequences of this process are considered to embrace at least two aspects: i) correlation between antimicrobial and membranotropic properties of this protein, and ii) lipid-mediated Lz amyloidogenesis. The mechanisms underlying the lipid-assisted protein fibrillogenesis and membrane disruption exerted by Lz in bacterial cells are believed to be similar. The present investigation was undertaken to gain further insight into Lz-lipid interactions and explore the routes by which Lz exerts its antimicrobial and amyloidogenic actions. Binding and Förster resonance energy transfer studies revealed that upon increasing the content of anionic lipids in lipid vesicles, Lz forms aggregates in a membrane environment. Total internal reflection fluorescence microscopy and pyrene excimerization reaction were employed to study the effect of Lz on the structural and dynamic properties of lipid bilayers. It was found that Lz induces lipid demixing and reduction of bilayer free volume, the magnitude of this effect being much more pronounced for oligomeric protein.

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The Human LL-37(17-29) Antimicrobial Peptide Reveals a Functional Supramolecular Nanostructure

10.1101/2020.02.04.933432 ◽

2020 ◽

Cited By ~ 2

Author(s):

Yizhaq Engelberg ◽

Meytal Landau

Keyword(s):

Lipid Bilayers ◽

Self Assembly ◽

Amyloid Fibrils ◽

Biological Activities ◽

Building Blocks ◽

Bacterial Cells ◽

Bacterial Membranes ◽

Self Assembling ◽

Wide Ribbon ◽

Wide Range

Protein fibrils that perform biological activities present attractive biomaterials. Here we demonstrate, by crystal structures, the self-assembly of the antibacterial human LL-37 active core (residues 17-29) into a stable structure of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-3717-29 correspondingly formed wide, ribbon-like, thermostable fibrils in solution, which co-localized with bacterial cells, and structure-guided mutagenesis analyses supported the role of self-assembly in antibacterial activity. LL-3717-29 resembled, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This suggests helical, self-assembling, basic building blocks across kingdoms of life and point to potential structural mimicry mechanisms. The findings offer a scaffold for functional and durable nanostructures for a wide range of medical and technological applications.

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Spectroscopy of model-membrane liposome-protein systems: complementarity of linear dichroism, circular dichroism, fluorescence and SERS

Emerging Topics in Life Sciences ◽

10.1042/etls20200354 ◽

2021 ◽

Author(s):

Anastasiia Tukova ◽

Alison Rodger

Keyword(s):

Circular Dichroism ◽

Lipid Bilayers ◽

Resonance Energy ◽

Lipid Vesicles ◽

Linear Dichroism ◽

Surface Enhanced Raman Spectroscopy ◽

Cellular Systems ◽

Spectroscopic Techniques ◽

Wide Range ◽

Circular Dichroïsm

A range of membrane models have been developed to study components of cellular systems. Lipid vesicles or liposomes are one such artificial membrane model which mimics many properties of the biological system: they are lipid bilayers composed of one or more lipids to which other molecules can associate. Liposomes are thus ideal to study the roles of cellular lipids and their interactions with other membrane components to understand a wide range of cellular processes including membrane disruption, membrane transport and catalytic activity. Although liposomes are much simpler than cellular membranes, they are still challenging to study and a variety of complementary techniques are needed. In this review article, we consider several currently used analytical methods for spectroscopic measurements of unilamellar liposomes and their interaction with proteins and peptides. Among the variety of spectroscopic techniques seeing increasing application, we have chosen to discuss: fluorescence based techniques such as FRET (fluorescence resonance energy transfer) and FRAP (fluorescence recovery after photobleaching), that are used to identify localisation and dynamics of molecules in the membrane; circular dichroism (CD) and linear dichroism (LD) for conformational and orientation changes of proteins on membrane binding; and SERS (Surface Enhanced Raman Spectroscopy) as a rapidly developing ultrasensitive technique for site-selective molecular characterisation. The review contains brief theoretical basics of the listed techniques and recent examples of their successful applications for membrane studies.

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Partitioning of semisynthetic lipidated N-Ras in lipid raft nanodomains determined by FRET to lipid domain markers

10.1101/221382 ◽

2017 ◽

Author(s):

Anna K. Shishina ◽

Elizaveta A. Kovrigina ◽

Azamat R. Galiakhmetov ◽

Rajendra Rathore ◽

Evgenii L. Kovrigin

Keyword(s):

Lipid Bilayers ◽

Membrane Surface ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Lipid Vesicles ◽

Lipid Domain ◽

Associated Proteins ◽

Liquid Ordered ◽

Raft Domains

ABSTRACTCellular membranes are heterogeneous planar lipid bilayers displaying lateral phase separation with the nanometer-scale liquid-ordered phase (aka “lipid rafts” or Lo) surrounded by the liquid-disordered phase (Ld). Many membrane-associated proteins were found to stably integrate in the rafts, which is critical for their biological function. Isoforms H and N of Ras GTPase possess a unique ability to switch their lipid domain preference depending on the type of bound guanine nucleotide (GDP or GTP). This behavior, however, has never been reproducedin vitroin model bilayers with recombinant proteins, and therefore has been attributed to action of other proteins binding Ras at the membrane surface. In this paper, we report the observation of the nucleotide-dependent switch of lipid domain preferences of the semisynthetic lipidated N-Ras in raft lipid vesiclesin the absence of other proteins. To detect segregation of Ras molecules in raft and disordered lipid domains, we measured Förster Resonance Energy Transfer (FRET) between the donor fluorophore, mant, attached to the protein-bound guanine nucleotides, and the acceptor, rhodamine-conjugated lipid, localized to the liquid-disordered domains. We demonstrated that N-Ras preferentially populated raft domains when bound to mant-GDP, while losing preference for rafts when it was associated with a GTP mimic, mant-GppNHp. At the same time, the isolated lipidated C-terminal peptide of N-Ras was found localized outside of the liquid-ordered rafts, most likely—in the bulk disordered lipid.

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Quantitative single molecule FRET efficiencies using TIRF microscopy

Faraday Discussions ◽

10.1039/c5fd00100e ◽

2015 ◽

Vol 184 ◽

pp. 131-142 ◽

Cited By ~ 8

Author(s):

Lasse L. Hildebrandt ◽

Søren Preus ◽

Victoria Birkedal

Keyword(s):

Single Molecule ◽

Structural Information ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Molecular Complexes ◽

Distance Information ◽

Single Molecule Level ◽

Single Molecule Fret ◽

Wide Range ◽

Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

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Binding of Citreoviridin to Human Serum Albumin: Multispectroscopic and Molecular Docking

BioMed Research International ◽

10.1155/2015/162391 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 10

Author(s):

Haifeng Hou ◽

Xiaolan Qu ◽

Yuqin Li ◽

Yueyue Kong ◽

Baoxiu Jia ◽

...

Keyword(s):

Molecular Modeling ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Animal Health ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Apparent Distance ◽

Wide Range ◽

Transfer Method

Citreoviridin (CIT), a mycotoxin produced byPenicillium citreonigrum,is a common contaminant of wide range of agriproducts and detrimental to human and animal health. In this study, the interaction of CIT with human serum albumin (HSA) is researched by steady-state fluorescence, ultraviolet-visible (UV-Vis) absorption, circular dichroism (CD) methods, and molecular modeling. The association constants, binding site numbers, and corresponding thermodynamic parameters are used to investigate the quenching mechanism. The alternations of HSA secondary structure in the presence of CIT are demonstrated with UV-Vis, synchronous fluorescence, and CD spectra. The molecular modeling results reveal that CIT can bind with hydrophobic pocket of HSA with hydrophobic and hydrogen bond force. Moreover, an apparent distance of 3.25 nm between Trp214 and CIT is obtained via fluorescence resonance energy transfer method.

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A glue for heterochromatin maintenance

The Journal of Cell Biology ◽

10.1083/jcb.200502154 ◽

2005 ◽

Vol 170 (4) ◽

pp. 537-549 ◽

Cited By ~ 51

Author(s):

Ilke M. Krouwels ◽

Karien Wiesmeijer ◽

Tsion E. Abraham ◽

Chris Molenaar ◽

Nico P. Verwoerd ◽

...

Keyword(s):

Fluorescence Recovery After Photobleaching ◽

Dynamic Properties ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Pericentromeric Heterochromatin ◽

Deletion Mutants ◽

Set Domain ◽

Heterochromatin Protein 1 ◽

Heterochromatin Protein ◽

Stable Component

Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721–725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719–721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.

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Methods used to study the oligomeric structure of G-protein-coupled receptors

Bioscience Reports ◽

10.1042/bsr20160547 ◽

2017 ◽

Vol 37 (2) ◽

Cited By ~ 32

Author(s):

Hui Guo ◽

Su An ◽

Richard Ward ◽

Yang Yang ◽

Ying Liu ◽

...

Keyword(s):

Energy Transfer ◽

G Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

G Protein Coupled Receptors ◽

Experimental Techniques ◽

Distribution Analysis ◽

Wide Range ◽

And Function ◽

G Protein Coupled

G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native PAGE (BN-PAGE), co-immunoprecipitation (Co-IP) and protein-fragment complementation assays (PCAs), those based upon resonance energy transfer, FRET, time-resolved FRET (TR-FRET), FRET spectrometry and bioluminescence resonance energy transfer (BRET). Those based upon microscopy such as FRAP, total internal reflection fluorescence microscopy (TIRFM), spatial intensity distribution analysis (SpIDA) and various single molecule imaging techniques. Finally with the solution of a growing number of crystal structures, X-ray crystallography must be acknowledged as an important source of discovery in this field. A different, but in many ways complementary approach to the use of more traditional experimental techniques, are those involving computational methods that possess obvious merit in the study of the dynamics of oligomer formation and function. Here, we summarize the latest developments that have been made in the methods used to study GPCR oligomerization and give an overview of their application.

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DNA Duplexes with Hydrophobic Modifications Inhibit Fusion between HIV-1 and Cell Membranes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00758-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4963-4970 ◽

Cited By ~ 8

Author(s):

Liang Xu ◽

Lifeng Cai ◽

Xueliang Chen ◽

Xifeng Jiang ◽

Huihui Chong ◽

...

Keyword(s):

Lipid Bilayers ◽

Cell Membranes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

New Drugs ◽

Heptad Repeat ◽

Dna Duplexes ◽

Dna Duplex ◽

Modified Dna ◽

Hiv 1

ABSTRACTDiscovery of new drugs for the treatment of AIDS typically possessing unique structures associated with novel mechanisms of action has been of great importance due to the quick drug-resistant mutations of HIV-1 strains. The work presented in this report describes a novel class of DNA duplex-based HIV-1 fusion inhibitors. Hydrophobic groups were introduced into a DNA duplex skeleton either at one end, at both ends, or in the middle. These modified DNA duplexes inhibited fusion between HIV-1 and human cell membranes at micro- or submicromolar concentrations. Respective inhibitors adopted an aptamer pattern instead of a base-pairing interaction pattern. Structure-activity relationship studies of the respective DNA duplexes showed that the rigid and negatively charged DNA skeletons, in addition to the presence of hydrophobic groups, were crucial to the anti-HIV-1 activity of these compounds. A fluorescent resonance energy transfer (FRET)-based inhibitory assay showed that these duplex inhibitors interacted with the primary pocket in the gp41 N-terminal heptad repeat (NHR) instead of interacting with the lipid bilayers.

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Versatility of Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assays for Biologics Drug Discovery

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057114557464 ◽

2014 ◽

Vol 20 (4) ◽

pp. 508-518 ◽

Cited By ~ 5

Author(s):

Christine J. Rossant ◽

Carl Matthews ◽

Frances Neal ◽

Caroline Colley ◽

Matthew J. Gardener ◽

...

Keyword(s):

Energy Transfer ◽

Drug Discovery ◽

Fluorescence Resonance Energy Transfer ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Donor And Acceptor ◽

Wide Range ◽

Fluorescence Resonance

Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability of donor and acceptor fluorophore-conjugated reagents for detection of multiple tags or fusion proteins. The time-resolved component of the technology reduces potential assay interference, allowing screening of a range of different crude sample types derived from the bacterial or mammalian cell expression systems often used for antibody discovery projects. Here we describe the successful application of this technology across multiple projects targeting soluble proteins and demonstrate how it has provided key information for the isolation of potential therapeutic antibodies with the desired activity profile.

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