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

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.

scholarly journals UV-CD12: synchrotron radiation circular dichroism beamline at ANKA

2015 ◽  
Vol 22 (3) ◽  
pp. 844-852 ◽  
Author(s):  
Jochen Bürck ◽  
Siegmar Roth ◽  
Dirk Windisch ◽  
Parvesh Wadhwani ◽  
David Moss ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Synchrotron Radiation ◽  
Lipid Bilayers ◽  
Spectral Range ◽  
Signal To Noise Ratio ◽  
Linear Dichroism ◽  
Water Soluble ◽  
Protein Films ◽  
Hydrated Protein ◽  
Circular Dichroïsm

Synchrotron radiation circular dichroism (SRCD) is a rapidly growing technique for structure analysis of proteins and other chiral biomaterials. UV-CD12 is a high-flux SRCD beamline installed at the ANKA synchrotron, to which it had been transferred after the closure of the SRS Daresbury. The beamline covers an extended vacuum-UV to near-UV spectral range and has been open for users since October 2011. The current end-station allows for temperature-controlled steady-state SRCD spectroscopy, including routine automated thermal scans of microlitre volumes of water-soluble proteins down to 170 nm. It offers an excellent signal-to-noise ratio over the whole accessible spectral range. The technique of oriented circular dichroism (OCD) was recently implemented for determining the membrane alignment of α-helical peptides and proteins in macroscopically oriented lipid bilayers as mimics of cellular membranes. It offers improved spectral quality <200 nm compared with an OCD setup adapted to a bench-top instrument, and accelerated data collection by a factor of ∼3. In addition, it permits investigations of low hydrated protein films down to 130 nm using a rotatable sample cell that avoids linear dichroism artifacts.

scholarly journals Modulation of physiological and pathological activities of lysozyme by biological membranes

2012 ◽  
Vol 17 (3) ◽  
Author(s):  
Valeriya Trusova
Keyword(s):  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Dynamic Properties ◽  
Biological Activities ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Lipid Vesicles ◽  
Bacterial Cells ◽  
Oligomeric Protein ◽  
Wide Range

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.

Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica

2019 ◽  
Vol 26 (6) ◽  
pp. 435-448
Author(s):  
Priyanka Biswas ◽  
Dillip K. Sahu ◽  
Kalyanasis Sahu ◽  
Rajat Banerjee
Keyword(s):  
Circular Dichroism ◽  
Steady State ◽  
Entamoeba Histolytica ◽  
Protein Concentration ◽  
Trna Synthetase ◽  
Solvent Exposure ◽  
Steady State Fluorescence ◽  
State Process ◽  
Wide Range ◽  
Circular Dichroïsm

Background: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. Objective: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. Methods: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. Results: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. Conclusion: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two – state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

Conformation of membrane-bound proteins revealed by vacuum-ultraviolet circular-dichroism and linear-dichroism spectroscopy

2016 ◽  
Vol 84 (3) ◽  
pp. 349-359 ◽  
Author(s):  
Koichi Matsuo ◽  
Yasuyuki Maki ◽  
Hirofumi Namatame ◽  
Masaki Taniguchi ◽  
Kunihiko Gekko
Keyword(s):  
Circular Dichroism ◽  
Vacuum Ultraviolet ◽  
Linear Dichroism ◽  
Membrane Bound ◽  
Circular Dichroïsm

scholarly journals Absorption and Circular Dichroism Spectra of Molecular Aggregates with the Full Cumulant Expansion.

2020 ◽  
Author(s):  
Lorenzo Cupellini ◽  
Filippo Lipparini ◽  
Jianshu Cao
Keyword(s):  
Circular Dichroism ◽  
Purple Bacteria ◽  
Spectroscopic Techniques ◽  
Molecular Aggregates ◽  
Cumulant Expansion ◽  
Circular Dichroism Spectra ◽  
Linear Absorption ◽  
Redfield Theory ◽  
Vibronic Sideband ◽  
Circular Dichroïsm

The exciton Hamiltonian of multichromophoric aggregates can be probed by spectroscopic techniques such as linear absorption and circular dichroism. In order to compare calculated Hamiltonians to experiments, a lineshape theory is needed, which takes into account the coupling of the excitons with inter- and intramolecular vibrations. This coupling is normally introduced in a perturbative way through the cumulant expansion formalism, and further approximated by assuming a Markovian exciton dynamics, for example with the modified Redfield theory.<br><br>Here we present an implementation of the full cumulant expansion (FCE) formalism [Ma and Cao, <i>J. Chem. Phys.</i> <b>2015</b>, 142, 094106 ] to efficiently compute absorption and circular dichroism spectra of molecular aggregates beyond the Markov approximation, without restrictions on the form of the exciton-phonon coupling. By employing the LH2 system of purple bacteria as a challenging test case, we compare the FCE lineshapes with the Markovian lineshapes obtained with the modified Redfield theory, showing that the latter present a much poorer agreement with experiments. The FCE approach instead accurately describes the lineshapes, especially in the vibronic sideband of the B800 peak. We envision that the FCE approach will become a valuable tool for accurately comparing model exciton Hamiltonians with optical spectroscopy experiments.

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