scholarly journals Lipid vesicle fusion as a vehicle to study transmembrane protein interactions

2021 ◽  
Author(s):  
◽  
Bryan M. Lada
Keyword(s):  
Secondary Structure ◽  
Fluorescence Intensity ◽  
Lipid Vesicles ◽  
Vesicle Fusion ◽  
Spectroscopic Technique ◽  
Cationic Lipids ◽  
Lipid Vesicle ◽  
Aqueous Environment ◽  
Lipid Environment ◽  
Protein Enzyme

Alzheimer's disease, among other neurologically degenerative diseases, has been linked to protein-enzyme interactions that originate within the transmembrane domain of a cell. The lipid environment that houses these interactions lends difficulty to studying intramembrane interactions, often making for time consuming data analysis. Lengthy data interpretation on top of the rate in which protein-enzyme interactions take place creates a need for a method to overcome these obstacles. The use of lipid vesicle fusion to apply a margin of control over the time frame of interaction combined with deep probing spectroscopic techniques can minimize the interference of the lipid environment. Deep ultraviolent resonance Raman (dUVRR) is a vibrational spectroscopic technique that probes along the protein backbone that allows for removal of lipid environmental interference through background subtraction. Lipid vesicle fusion has been demonstrated by mixing lipid vesicles comprised of oppositely charged head groups (cationic 1,2-diaurroyl-sn-glycero-3-phospho-(1-rac-glycerol) (DLPG)) and anionic 1,2-dilauroyl-sn-glycero-3-ethylphocholine (12:0 EPC) or 1,2-dimyristoyl-sn-glycero-3-ethylphocholine (14:0 EPC)) of equivalent or varying aliphatic tail length, up to a 2-carbon difference. The fluorescent dye, 8-aminonapthalene 1,3,6-trisulfonic acid (ANTS), paired with the quencher, p-xylene-bis-pyridiumbromide (DPX), are separately encased in either DLPG or 12:0 EPC/14:0 EPC, respectively, in aqueous solution, and evidence of lipid vesicle fusion is provided by monitoring fluorescence intensity of ANTS as the two solutions are mixed, resulting in the closing proximity of ANTS and DPX observed as a decrease in fluorescence intensity. Additional evidence is provided by dynamic light scattering (DLS) measurements of both independent vesicle solutions and their mixture showing an increase in hydrodynamic radius (Rh). In addition, cohesion of similarly sized lipids is demonstrated, as DLPG (12-carbon chain) fails to fuse with cationic lipids of chain length 16 carbons or longer. Circular dichroism (CD) is a spectroscopic technique that uses left and right-handed polarized light to obtain the overall secondary structure of proteins. Shown is the use of CD to probe the secondary structure and the changes incurred on PolyLA7 (PLA7), a model ?-helical peptide when placed in a transmembrane or hydrophobic environment, through a change in the lipid environment. PLA7 was inserted in DLPG lipid vesicles and then mixed in solution with lipid vesicles comprised of 14:0 EPC. CD spectra were obtained pre and post vesicle fusion, demonstrating the use of lipid fusion as a means to combine membrane embedded proteins of interest while still being able to observe changes that take place. Finally, we propose an on-demand lipid fusion system in which two separate lipid vesicles could be co-suspended in solution and then chemically or photonically induced to fuse. A titration was performed to obtain the pKa of a synthesized pH inducible cationic lipid (pHiCL). The pHiCL is a dipicolylamine with an attached 12-carbon aliphatic tail. The pHiCL was titrated while suspended in an aqueous environment and while inserted into a lipid vesicle comprised of DLPC, a net neutral lipid also with a 12-carbon length aliphatic tail. The pHiCL will be the first component of a two-part system in which a photoacid (PA) will be used to protonate the pHiCL in solution giving rise to cationic and anionic surfaced lipid vesicles causing vesicle fusion to occur.

scholarly journals A nano flow cytometer for single lipid vesicle analysis

Lab on a Chip ◽  
2017 ◽  
Vol 17 (5) ◽  
pp. 830-841 ◽  
Author(s):  
Remo Friedrich ◽  
Stephan Block ◽  
Mohammadreza Alizadehheidari ◽  
Susanne Heider ◽  
Joachim Fritzsche ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Lipid Vesicles ◽  
Flow Cytometer ◽  
Lipid Vesicle ◽  
Single Vesicle

A nano flow cytometer to quantify lipid vesicles and to analyse fluorescence intensity on a single vesicle level is presented.

scholarly journals Fast Simulation of Lipid Vesicle Deformation Using Spherical Harmonic Approximation

2016 ◽  
Vol 21 (1) ◽  
pp. 40-64
Author(s):  
Michael Mikucki ◽  
Yongcheng Zhou
Keyword(s):  
Harmonic Functions ◽  
Degrees Of Freedom ◽  
Finite Difference Methods ◽  
Harmonic Approximation ◽  
Lipid Vesicles ◽  
Membrane Curvature ◽  
Lipid Vesicle ◽  
Nonlinear Conjugate Gradient Method ◽  
Surface Harmonic ◽  
Curvature Energy

AbstractLipid vesicles appear ubiquitously in biological systems. Understanding how the mechanical and intermolecular interactions deform vesicle membranes is a fundamental question in biophysics. In this article we develop a fast algorithm to compute the surface configurations of lipid vesicles by introducing surface harmonic functions to approximate themembrane surface. This parameterization allows an analytical computation of the membrane curvature energy and its gradient for the efficient minimization of the curvature energy using a nonlinear conjugate gradient method. Our approach drastically reduces the degrees of freedom for approximating the membrane surfaces compared to the previously developed finite element and finite difference methods. Vesicle deformations with a reduced volume larger than 0.65 can be well approximated by using as small as 49 surface harmonic functions. The method thus has a great potential to reduce the computational expense of tracking multiple vesicles which deform for their interaction with external fields.

scholarly journals Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F1F0-ATP synthase

2017 ◽  
Vol 114 (43) ◽  
pp. 11291-11296 ◽  
Author(s):  
Víctor G. Almendro-Vedia ◽  
Paolo Natale ◽  
Michael Mell ◽  
Stephanie Bonneau ◽  
Francisco Monroy ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Lipid Membranes ◽  
Lipid Vesicles ◽  
Proton Pumping ◽  
Collective Effects ◽  
Coupling Mechanism ◽  
Nonequilibrium Fluctuations ◽  
Membrane Fluctuations ◽  
Lipid Environment ◽  
Curved Membrane

ATP synthase is a rotating membrane protein that synthesizes ATP through proton-pumping activity across the membrane. To unveil the mechanical impact of this molecular active pump on the bending properties of its lipid environment, we have functionally reconstituted the ATP synthase in giant unilamellar vesicles and tracked the membrane fluctuations by means of flickering spectroscopy. We find that ATP synthase rotates at a frequency of about 20 Hz, promoting large nonequilibrium deformations at discrete hot spots in lipid vesicles and thus inducing an overall membrane softening. The enhanced nonequilibrium fluctuations are compatible with an accumulation of active proteins at highly curved membrane sites through a curvature−protein coupling mechanism that supports the emergence of collective effects of rotating ATP synthases in lipid membranes.

scholarly journals Refolding of urea-denatured adenylate kinase

1998 ◽  
Vol 333 (2) ◽  
pp. 401-405 ◽  
Author(s):  
Hong-jie ZHANG ◽  
Xiang-rong SHENG ◽  
Xian-ming PAN ◽  
Jun-mei ZHOU
Keyword(s):  
Secondary Structure ◽  
Rate Constant ◽  
Fluorescence Intensity ◽  
Adenylate Kinase ◽  
Surface Hydrophobicity ◽  
Native Structure ◽  
Fast Process ◽  
Ans Binding ◽  
First Order ◽  
Partially Folded Intermediate

The refolding of urea-denatured adenylate kinase (EC 2.7.4.3) has been followed by formation of the secondary structure, change of surface hydrophobicity and recovery of catalytic activity. During refolding of adenylate kinase with a 20–80-fold dilution of 4 M urea-denatured enzyme at 10 °C, the formation of the secondary structure is a fast process with a rate constant of > 0.16 s-1. Transient enhancement of the 8-anilino-1-naphthalenesulphonate (ANS) fluorescence intensity is followed by a fluorescence decrease to the level equal to the value characteristic of native enzyme. The desorption of ANS binding fluorescence is relatively slow and can be fitted to a first order reaction with a rate constant of 0.004 s-1 when the ANS is present in the dilution buffer. The desorption of ANS-binding fluorescence is accelerated in the presence of nucleotide substrates. The rate constants are increased to 0.049, 0.029, 0.028 and 0.029 s-1 in the presence of 1 mM AMP, MgATP, ATP and ADP respectively. The refolding rate constant calculated from the initial fluorescence intensity after mixing ANS with protein at different refolding intervals is 0.016 s-1, which is faster than those obtained when ANS is present throughout the refolding process, indicating that the binding of ANS with a partially folded intermediate retards its further refolding to its native structure. The reactivation rate is even faster than the rates of refolding monitored in the absence of substrates, showing that the refolding is accelerated in the presence of the substrates. A possible refolding pathway and the accelerating effect of substrates are discussed.

scholarly journals Characterizing the lipid-protein interface of the human serotonin transporter by crosslinking mass spectrometry

2020 ◽  
Author(s):  
A.G. DeMarco ◽  
N.A. Ferraro ◽  
K. Sweigard ◽  
M. Cascio
Keyword(s):  
Mass Spectrometry ◽  
Serotonin Transporter ◽  
Lipid Vesicles ◽  
Human Cell Line ◽  
Depression And Anxiety ◽  
Disease States ◽  
Lipid Environment ◽  
Carboxy Terminal ◽  
Lipid Accessibility ◽  
Apo State

ABSTRACTAltered serotonin (5-HT) levels contribute to disease states such as depression and anxiety. Synaptic serotonin concentration is partially regulated by the serotonin transporter (SERT), making this transporter an important therapeutic target. This study seeks to examine the lipid accessible domains of hSERT to provide critical information regarding the apo-state of this transporter in a lipid environment. Recombinant hSERT was inducibly expressed in a human cell line. Solubilized SERT was purified by affinity chromatography using a FLAG Tag and reconstituted into mixed lipid vesicles containing our photoactivatable lipid probe. The lipid-accessible domains of the reconstituted transporter in membranes in its apo-state were probed via photocrosslinking to azi-cholesterol followed by quadrupole time of flight liquid chromatography-mass spectrometry (QTOF-LC-MS). MS studies identified crosslinks in three transmembrane loops consistent with the known topology of SERT. Surprisingly, the amino- and carboxy-terminal domains were similarly crosslinked by cholesterol, suggesting that these regions may also be intimately associated with the lipid bilayer. The data presented herein assist in further refining our understanding of the topography of the apo-state of hSERT via analysis of lipid accessibility.

scholarly journals Folding Behaviour and Antibacterial Activity of Ionic Complementary Peptide EAK-16

2021 ◽  
pp. 122-130
Author(s):  
Abdul Majid ◽  
Farah Naz ◽  
Hatim Ali Jamro ◽  
Sham Lal ◽  
Inayatullah Soomro ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Neutral Lipids ◽  
Lipid Vesicles ◽  
Aqueous Environment ◽  
Antibacterial Efficacy ◽  
Minimum Concentration ◽  
Membrane Perturbation ◽  
Structural Conformation ◽  
Membrane Interfaces ◽  
Calcein Leakage

Aim: A major challenge in the development of new antibiotics is the biocompatibility within biological environment. Ionic complementary peptide (EAK-16) from amyloid protein, have the ability to adopt secondary structure conformation at membrane interfaces. This study aimed to investigate the effect of membrane on EAK-16 peptide folding and their antibacterial applications. Methodology: We studied secondary structural conformation of EAK-16 using circular dichroism (CD) spectroscopy in an aqueous environment and at membrane bilayers interfaces. Initially, the antibacterial efficacy was investigated against both Gram-positive and Gram-negative bacteria. Membrane mimicking models were synthesised with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS) lipid vesicles using calcein leakage assay. Results: EAK-16 showed transition in secondary structural conformation. In aqueous environment, it was predominantly β-sheets and at membrane interfaces, it was mainly α-helical. EAK-16 peptide was highly active against bacteria (at minimum concentration applied) and membrane leakage was found to be > 60%. This effect was confirmed with both anionic lipids (DMPS) and neutral lipids (DMPC). The helical transition of EAK-16 could be a major factor to disrupt the membrane and bacterial death Conclusion: The secondary structural conformation and calcein leakage data suggest that EAK-16 has potential to kill bacteria by adopting helical tilted conformation and membrane perturbation via lysis. This study revealed structure-function relationship of peptide and lipid bilayers to further investigate the mode of pore formation and mode of action of EAK-16 in membrane perturbation and antibacterial efficacy.

Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish

2005 ◽  
Vol 288 (5) ◽  
pp. R1264-R1272 ◽  
Author(s):  
Samantha J. Richardson ◽  
Julie A. Monk ◽  
Caroline A. Shepherdley ◽  
Lars O. E. Ebbesson ◽  
Frank Sin ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Thyroid Hormones ◽  
Sea Bream ◽  
Aqueous Environment ◽  
Critical Periods ◽  
Developmentally Regulated ◽  
Thyroxine Binding Globulin ◽  
Hormone Levels ◽  
Thyroid Hormone Levels ◽  
Lipid Environment

Thyroid hormones are essential for vertebrate development. There is a characteristic rise in thyroid hormone levels in blood during critical periods of thyroid hormone-regulated development. Thyroid hormones are lipophilic compounds, which readily partition from an aqueous environment into a lipid environment. Thyroid hormone distributor proteins are required to ensure adequate distribution of thyroid hormones, throughout the aqueous environment of the blood, and to counteract the avid partitioning of thyroid hormones into the lipid environment of cell membranes. In human blood, these proteins are albumin, transthyretin and thyroxine-binding globulin. We analyzed the developmental profile of thyroid hormone distributor proteins in serum from a representative of each order of marsupials ( M. eugenii; S.crassicaudata), a reptile ( C. porosus), in two species of salmonoid fishes ( S. salar; O. tshawytsch), and throughout a calendar year for sea bream ( S. aurata). We demonstrated that during development, these animals have a thyroid hormone distributor protein present in their blood which is not present in the adult blood. At least in mammals, this additional protein has higher affinity for thyroid hormones than the thyroid hormone distributor proteins in the blood of the adult. In fish, reptile and polyprotodont marsupial, this protein was transthyretin. In a diprotodont marsupial, it was thyroxine-binding globulin. We propose an hypothesis that an augmented thyroid hormone distributor protein network contributes to the rise in total thyroid hormone levels in the blood during development.

Deformation and rupture of lipid vesicles in the strong shear flow generated by ultrasound-driven microbubbles

2008 ◽  
Vol 464 (2095) ◽  
pp. 1781-1800 ◽  
Author(s):  
Philippe Marmottant ◽  
Thierry Biben ◽  
Sascha Hilgenfeldt
Keyword(s):  
Shear Flow ◽  
Acoustic Streaming ◽  
Three Dimensional ◽  
Spherical Shape ◽  
Lipid Vesicles ◽  
Boundary Integral ◽  
Elastic Response ◽  
Lipid Vesicle ◽  
Strong Shear ◽  
Vesicle Rupture

Considering the elastic response of the membrane of a lipid vesicle (artificial cell) in an arbitrary three-dimensional shear flow, we derive analytical predictions of vesicle shape and membrane tension for vesicles close to a spherical shape. Large amplitude deviations from sphericity are described using boundary integral numerical simulations. Two possible modes of vesicle rupture are found and compared favourably with experiments: (i) for large enough shear rates the tension locally exceeds a rupture threshold and a pore opens at the waist of the vesicle and (ii) for large elongations the local tension becomes negative, leading to buckling and tip formation near a pole of the vesicle. We experimentally check these predictions in the case of strong acoustic streaming flow generated near ultrasound-driven microbubbles, such as those used in medical applications.

Lipid Vesicle Fusion on μCP Patterned Self-Assembled Monolayers:  Effect of Pattern Geometry on Bilayer Formation

Langmuir ◽  
2002 ◽  
Vol 18 (8) ◽  
pp. 3176-3180 ◽  
Author(s):  
A. Toby A. Jenkins ◽  
Richard J. Bushby ◽  
Stephen D. Evans ◽  
Wolfgang Knoll ◽  
Andreas Offenhäusser ◽  
...  
Keyword(s):  
Vesicle Fusion ◽  
Self Assembled Monolayers ◽  
Lipid Vesicle ◽  
Assembled Monolayers ◽  
Self Assembled ◽  
Bilayer Formation
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