共查询到20条相似文献,搜索用时 625 毫秒
1.
Dr. Thirupathi Ravula Dr. Sudheer Kumar Ramadugu Giacomo Di Mauro Prof. Ayyalusamy Ramamoorthy 《Angewandte Chemie (International ed. in English)》2017,56(38):11466-11470
Polymer‐based nanodiscs are valuable tools in biomedical research that can offer a detergent‐free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6 kDa SMA‐based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer‐based nanodiscs are characterized by light scattering, NMR, FT‐IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub‐micrometer diameter, can be produced by varying the lipid‐to‐polymer ratio. The small‐size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic‐alignment of macro‐nanodiscs (diameter of > ca. 40 nm) can be exploited for solid‐state NMR studies on membrane proteins. 相似文献
2.
Thirupathi Ravula Nathaniel Z. Hardin Sudheer Kumar Ramadugu Sarah J. Cox Prof. Ayyalusamy Ramamoorthy 《Angewandte Chemie (International ed. in English)》2018,57(5):1342-1345
Polymer lipid nanodiscs are an invaluable system for structural and functional studies of membrane proteins in their near‐native environment. Despite the recent advances in the development and usage of polymer lipid nanodisc systems, lack of control over size and poor tolerance to pH and divalent metal ions are major limitations for further applications. A facile modification of a low‐molecular‐weight styrene maleic acid copolymer is demonstrated to form monodispersed lipid bilayer nanodiscs that show ultra‐stability towards divalent metal ion concentration over a pH range of 2.5 to 10. The macro‐nanodiscs (>20 nm diameter) show magnetic alignment properties that can be exploited for high‐resolution structural studies of membrane proteins and amyloid proteins using solid‐state NMR techniques. The new polymer, SMA‐QA, nanodisc is a robust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems. 相似文献
3.
Thirupathi Ravula Ayyalusamy Ramamoorthy 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2019,131(42):15067-15070
Experimentally measured residual dipolar couplings (RDCs) are highly valuable for atomic‐resolution structural and dynamic studies of molecular systems ranging from small molecules to large proteins by solution NMR spectroscopy. Here we demonstrate the first use of magnetic‐alignment behavior of lyotropic liquid‐crystalline polymer macro‐nanodiscs (>20 nm in diameter) as a novel alignment medium for the measurement of RDCs using high‐resolution NMR. The easy preparation of macro‐nanodiscs, their high stability against pH changes and the presence of divalent metal ions, and their high homogeneity make them an efficient tool to investigate a wide range of molecular systems including natural products, proteins, and RNA. 相似文献
4.
《Angewandte Chemie (International ed. in English)》2017,56(9):2508-2512
The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane‐mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co‐polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high‐resolution solid‐state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD. 相似文献
5.
Nathaniel Z. Hardin Voj
Kocman Giacomo M. Di Mauro Thirupathi Ravula Ayyalusamy Ramamoorthy 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2019,131(48):17406-17410
Paramagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T1) for rapid data acquisition in NMR structural studies. Consequently, there is significant interest in novel paramagnetic labels for enhanced NMR studies on biomolecules. Herein, we report the synthesis and characterization of a modified poly(styrene‐co‐maleic acid) polymer which forms nanodiscs while showing the ability to chelate metal ions. Cu2+‐chelated nanodiscs are demonstrated to reduce the T1 of protons for both polymer and lipid‐nanodisc components. The chelated nanodiscs also decrease the proton T1 values for a water‐soluble DNA G‐quadruplex. These results suggest that polymer nanodiscs functionalized with paramagnetic tags can be used to speed‐up data acquisition from lipid bilayer samples and also to provide structural information from water‐soluble biomolecules. 相似文献
6.
《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2018,130(13):3449-3453
Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide‐based lipid nanodiscs to “trap” the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein‐induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane. 相似文献
7.
Probing the Lipid Annular Belt by Gas‐Phase Dissociation of Membrane Proteins in Nanodiscs
下载免费PDF全文
![点击此处可从《Angewandte Chemie (International ed. in English)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Dr. Michael T. Marty Kin Kuan Hoi Dr. Joseph Gault Prof. Carol V. Robinson 《Angewandte Chemie (International ed. in English)》2016,55(2):550-554
Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment. 相似文献
8.
《Angewandte Chemie (International ed. in English)》2017,56(7):1919-1924
Once removed from their natural environment, membrane proteins depend on membrane‐mimetic systems to retain their native structures and functions. To this end, lipid‐bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane‐protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl‐chain order, does not interfere with optical spectroscopy in the far‐UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations. 相似文献
9.
Structural Studies of Self‐Assembled Subviral Particles: Combining Cell‐Free Expression with 110 kHz MAS NMR Spectroscopy
下载免费PDF全文
![点击此处可从《Angewandte Chemie (International ed. in English)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Guillaume David Dr. Marie‐Laure Fogeron Maarten Schledorn Roland Montserret Uta Haselmann Susanne Penzel Dr. Aurélie Badillo Dr. Lauriane Lecoq Prof. Patrice André Prof. Michael Nassal Prof. Ralf Bartenschlager Prof. Beat H. Meier Dr. Anja Böckmann 《Angewandte Chemie (International ed. in English)》2018,57(17):4787-4791
Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native‐like lipid environment. Cell‐free protein synthesis and solid‐state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell‐free expression, and that the protein self‐assembles into subviral particles. Proton‐detected 2D NMR spectra recorded at a magic‐angle‐spinning frequency of 110 kHz on <500 μg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection. 相似文献
10.
Wadsäter M Simonsen JB Lauridsen T Tveten EG Naur P Bjørnholm T Wacklin H Mortensen K Arleth L Feidenhans'l R Cárdenas M 《Langmuir : the ACS journal of surfaces and colloids》2011,27(24):15065-15073
Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 ? with a surface coverage of 66 ± 4%. This layer is located about 15 ? below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 ?) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer. 相似文献
11.
《Macromolecular bioscience》2017,17(9)
A hybrid hydrogel composed of solid lipid nanoparticles (LNPs) entrapped within chemically cross‐linked carboxymethylcellulose (CMC) is developed to achieve localized and sustained release of lipophilic drugs. The analysis of LNP stability as well as the hydrogel swelling and mechanical properties confirm the successful incorporation of particles up to a concentration of 50% w/wCMC. The initial LNP release rate can be prolonged by increasing the particle diameter from 50 to 120 nm, while the amount of long‐term release can be adjusted by tailoring the particle surface charge or the cross‐linking density of the polymer. After 30 d, 58% of 50 nm diameter negatively charged LNPs escape from the matrix while only 17% of positively charged nanoparticles are released from materials with intermediate cross‐linking density. A mathematical diffusion model based on Fick's second law is efficient to predict the diffusion of the particles from the hydrogels. 相似文献
12.
Manuela Zanetti Devid Maniglio Cristian Fasoli Marco Pola Elisa Borga Claudio Corradi Mauro Dalla Serra Salvatore Iannotta Antonella Motta Tullio Toccoli 《Electroanalysis》2014,26(8):1653-1659
We report the fabrication of a microfluidic apparatus and the realization of a sensors based on PEDOT : PSS, a biocompatible semiconductor polymer used in substitution of standard electrodes for electrophysiological studies and for detection of nanopores in membrane. This gives the possibility to study the mechanisms of ions balance and molecular transport though cell membranes. In particular the apparatus is based on two chambers connected through an aperture in a PTFE sheet where lipid bilayer are formed using Montal‐Mueller method, and the pore‐forming proteins activity is detected by polymeric electrodes. This methodology could be applied to examine different membrane proteins for the purpose of biosensing, drug screening and nanopore technologies. 相似文献
13.
《Angewandte Chemie (International ed. in English)》2017,56(1):380-383
Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility. 相似文献
14.
Conformation and Topology of Diacylglycerol Kinase in E.coli Membranes Revealed by Solid‐state NMR Spectroscopy
下载免费PDF全文
![点击此处可从《Angewandte Chemie (International ed. in English)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Yanke Chen Dr. Zhengfeng Zhang Xinqi Tang Jianping Li Prof. Clemens Glaubitz Prof. Jun Yang 《Angewandte Chemie (International ed. in English)》2014,53(22):5624-5628
Solid‐state NMR is a powerful tool for studying membrane proteins in a native‐like lipid environment. 3D magic angle spinning (MAS) NMR was employed to characterize the structure of E.coli diacylglycerol kinase (DAGK) reconstituted into its native E.coli lipid membranes. The secondary structure and topology of DAGK revealed by solid‐state NMR are different from those determined by solution‐state NMR and X‐ray crystallography. This study provides a good example for demonstrating the influence of membrane environments on the structure of membrane proteins. 相似文献
15.
《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2017,129(4):1007-1011
The direct reaction between copper nitrate, thymine‐1‐acetic acid, and 4,4′‐bipyridine in water leads to the formation of a blue colloid comprising uniform crystalline nanoribbons (length >1 μm; width ca. 150–185 nm; diameter ca. 15–60 nm) of a coordination polymer. The polymer displays a thymine‐based structure freely available for supramolecular interactions. These nanostructures show significant selective interaction with single‐stranded oligonucleotides based on adenine. Remarkably, they present low cell toxicity in three cell lines–despite the copper(II) content–and can be used as nanocarriers of oligonucleotides. These results suggest the potential of these types of nanostructures in several biological applications. 相似文献
16.
Michelle D. Farrelly Prof. Lisandra L. Martin Prof. San H. Thang 《Chemistry (Weinheim an der Bergstrasse, Germany)》2021,27(51):12922-12939
Membrane proteins (MPs) play a pivotal role in cellular function and are therefore predominant pharmaceutical targets. Although detailed understanding of MP structure and mechanistic activity is invaluable for rational drug design, challenges are associated with the purification and study of MPs. This review delves into the historical developments that became the prelude to currently available membrane mimetic technologies before shining a spotlight on polymer nanodiscs. These are soluble nanosized particles capable of encompassing MPs embedded in a phospholipid ring. The expanding range of reported amphipathic polymer nanodisc materials is presented and discussed in terms of their tolerance to different solution conditions and their nanodisc properties. Finally, the analytical scope of polymer nanodiscs is considered in both the demonstration of basic nanodisc parameters as well as in the elucidation of structures, lipid–protein interactions, and the functional mechanisms of reconstituted membrane proteins. The final emphasis is given to the unique benefits and applications demonstrated for native nanodiscs accessed through a detergent free process. 相似文献
17.
Reconstitution of the Cytb5–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy
下载免费PDF全文
![点击此处可从《Angewandte Chemie (International ed. in English)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Meng Zhang Rui Huang Dr. Rose Ackermann Dr. Sang‐Choul Im Prof. Lucy Waskell Prof. Anna Schwendeman Prof. Ayyalusamy Ramamoorthy 《Angewandte Chemie (International ed. in English)》2016,55(14):4497-4499
Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full‐length membrane‐bound P450 and its redox partner cytochrome b5 (cytb5) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane‐bound complex in model membranes renders high‐resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450–cytb5 complex in peptide‐based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450–cytb5 complex in the nanodisc. This is the first successful demonstration of a protein–protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane‐bound protein complexes. 相似文献
18.
Nanodiscs have become a leading technology to solubilize membrane proteins for biophysical, enzymatic, and structural investigations.
Nanodiscs are nanoscale, discoidal lipid bilayers surrounded by an amphipathic membrane scaffold protein (MSP) belt. A variety
of analytical tools has been applied to membrane proteins in nanodiscs, including several recent mass spectrometry studies.
Mass spectrometry of full-length proteins is an important technique for analyzing protein modifications, for structural studies,
and for identification of proteins present in binding assays. However, traditional matrix-assisted laser desorption/ionization-time-of-flight
(MALDI-TOF) mass spectrometry methods for analyzing full-length membrane proteins solubilized in nanodiscs are limited by
strong signal from the MSP belt and weak signal from the membrane protein inside the nanodisc. Herein, we show that an optimized
ultra-thin layer MALDI sample preparation technique dramatically enhances the membrane protein signal and nearly completely
eliminates the MSP signal. First-shot MALDI and MALDI imaging are used to characterize the spots formed by the ultra-thin
layer method. Furthermore, the membrane protein enhancement and MSP suppression are shown to be independent of the type of
membrane protein and are applicable to mixtures of membrane proteins in nanodiscs. 相似文献
19.
Yifei Qi Jumin Lee Jeffery B. Klauda Wonpil Im 《Journal of computational chemistry》2019,40(7):893-899
Nanodiscs are discoidal protein–lipid complexes that have wide applications in membrane protein studies. Modeling and simulation of nanodiscs are challenging due to the absence of structures of many membrane scaffold proteins (MSPs) that wrap around the membrane bilayer. We have developed CHARMM-GUI Nanodisc Builder ( http://www.charmm-gui.org/input/nanodisc ) to facilitate the setup of nanodisc simulation systems by modeling the MSPs with defined size and known structural features. A total of 11 different nanodiscs with a diameter from 80 to 180 Å are made available in both the all-atom CHARMM and two coarse-grained (PACE and Martini) force fields. The usage of the Nanodisc Builder is demonstrated with various simulation systems. The structures and dynamics of proteins and lipids in these systems were analyzed, showing similar behaviors to those from previous all-atom and coarse-grained nanodisc simulations. We expect the Nanodisc Builder to be a convenient and reliable tool for modeling and simulation of nanodisc systems. © 2019 Wiley Periodicals, Inc. 相似文献
20.
A Micromechanical Cantilever‐Based Liposome Biosensor for Characterization of Protein‐Membrane Interaction
下载免费PDF全文
![点击此处可从《Electroanalysis》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have newly evaluated the interaction of lipid membrane with two different proteins of lysozyme and carbonic anhydrase from bovine (CAB) using a micro cantilever‐based liposome biosensor with a new droplet‐sealing structure. Herein 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) liposomes are used as model lipid membrane and are immobilized on the surface of cantilever. The interaction of DPPC liposome with the target protein causes deflection of the micro‐cantilever, which can stably be detected by measuring the resistance change of the strain gauge. The resistance change dependent on time is used to evaluate the characteristic of liposome‐protein interaction. The resistance of the cantilever‐based biosensor increases monotonously with time in both of the two protein solutions. Especially, chronological resistance change depends markedly on both the concentration and species of target proteins. Finally, these results lead us to conclude that the cantilever‐based liposome biosensor with the droplet‐sealing structure facilitates the characterization of protein‐membrane interaction. It also means that this biosensor is a promising candidate device for label‐free detection of concentration and species of different target proteins. 相似文献