Restoration of Membrane Environments for Membrane Proteins for Structural and Functional Studies

2020 ◽  
pp. 145-159
Author(s):  
Liguo Wang
Keyword(s):  
Membrane Proteins ◽  
Functional Studies

scholarly journals Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Ekaitz Errasti-Murugarren ◽  
Paola Bartoccioni ◽  
Manuel Palacín
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Membrane ◽  
Protein Stabilization ◽  
New Drugs ◽  
Cell Physiology ◽  
Protein Preparation ◽  
Functional Studies ◽  
Membrane Protein Stability ◽  
Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

scholarly journals Nanodiscs for structural and functional studies of membrane proteins

2016 ◽  
Vol 23 (6) ◽  
pp. 481-486 ◽  
Author(s):  
Ilia G Denisov ◽  
Stephen G Sligar
Keyword(s):  
Membrane Proteins ◽  
Functional Studies

scholarly journals New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Gabriella Angiulli ◽  
Harveer Singh Dhupar ◽  
Hiroshi Suzuki ◽  
Irvinder Singh Wason ◽  
Franck Duong Van Hoa ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Structural Basis ◽  
New Approach ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Membrane Protein Complexes ◽  
Membrane Protein Reconstitution

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present ‘on-gradient’ reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional ‘on-bead’ approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

scholarly journals Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

2020 ◽  
Author(s):  
Fei Jin ◽  
Yao Wang ◽  
Mengqi Wang ◽  
Minxuan Sun ◽  
Motoyuki Hattori
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Size Exclusion Chromatography ◽  
Fluorescence Detection ◽  
Size Exclusion ◽  
Expression Screening ◽  
Functional Studies ◽  
Membrane Protein Expression ◽  
Exclusion Chromatography

AbstractMembrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

scholarly journals Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications

2015 ◽  
Vol 32 (5-8) ◽  
pp. 139-155 ◽  
Author(s):  
Kazumi Hiruma-Shimizu ◽  
Hiroki Shimizu ◽  
Gary S. Thompson ◽  
Arnout P. Kalverda ◽  
Simon G. Patching
Keyword(s):  
Membrane Proteins ◽  
Chemical Synthesis ◽  
Functional Studies

Nanodisc characterization by analytical ultracentrifugation

2017 ◽  
Vol 6 (1) ◽  
pp. 3-14 ◽  
Author(s):  
Sayaka Inagaki ◽  
Rodolfo Ghirlando
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Analytical Ultracentrifugation ◽  
Size Composition ◽  
Functional Studies ◽  
Lipid Environment ◽  
The Many ◽  
Sample Homogeneity ◽  
Simple Fashion

AbstractDue to their unique properties, tunable size, and ability to provide a near native lipid environment, nanodiscs have found widespread use for the structural and functional studies of reconstituted membrane proteins. They have also been developed, albeit in a few applications, for therapeutic and biomedical use. For these studies and applications, it is essential to characterize the nanodisc preparations in terms of their monodispersity, size, and composition, as these can influence the properties of the membrane protein of interest. Of the many biophysical methods utilized for the study and characterization of nanodiscs, we show that analytical ultracentrifugation is able to report on sample homogeneity, shape, size, composition, and membrane protein stoichiometry or oligomerization state in a direct and simple fashion. The method is truly versatile and does not require nanodisc modification or disassembly.

Absence of Myosin on Platelet Membranes Supported by Membrane Iodination, Immuno-Fluorescence and Functional Studies

1979 ◽  
Author(s):  
J.C. Mattson ◽  
W.J. Esselman ◽  
M.L. Schwarz ◽  
C.A. Zuiches
Keyword(s):  
Membrane Proteins ◽  
Protein Complex ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Clot Retraction ◽  
Platelet Surface ◽  
Functional Studies ◽  
Sds Page ◽  
Formalin Fixed

Immunofluorescence and functional studies were performed utilizing the immunoglobulin fraction of antisera raised against chromatographically purified human platelet myosin. When non-permeable, formalin fixed platelets were used, no FITC staining of the platelet membrane occurred indicating the myosin is not present on the platelet surface. When similar studies were performed on formalin fixed platelets using antibodies raised against the contractile protein complex, thrombosthenin, membrane fluorescence occurred. Autoradiographs of SDS-PAGE gels of the immune precipitate produced by reacting 125I labeled human platelet thrombosthenin with antithrombosthenin demonstrated that anti-thrombosthenin antisera was capable of reacting with at least three iodinated proteins In addition to myosin. 125I lactoperoxidase catalyzed labeling of the external membrane proteins of resting and ADP stimulated platelets indicated no external labeling of myosin although actin appeared to be labeled. In functional studies, incubation of human platelets with antimyosin did not produce aggregation nor did it Inhibit subsequent aggregation by ADP, collagen or epinephrine. Similarly, treatment of intact platelets with antimyosin did not cause Inhibition of clot retraction. These studies support the thesis that myosin is not localized on the external platelet membrane.

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