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.

scholarly journals Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Erik Henrich ◽  
Oliver Peetz ◽  
Christopher Hein ◽  
Aisha Laguerre ◽  
Beate Hoffmann ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Synthetic Biology ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Molecular Mechanisms ◽  
Protein Oligomerization ◽  
Protein Assemblies ◽  
Oligomeric State ◽  
Lipid Environment

Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.

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 Characterization of Outer Membrane Proteins in Chlamydia trachomatis LGV Serovar L2

2001 ◽  
Vol 183 (8) ◽  
pp. 2686-2690 ◽  
Author(s):  
Regina J. Tanzer ◽  
Thomas P. Hatch
Keyword(s):  
Chlamydia Trachomatis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Major Outer Membrane Protein ◽  
Developmental Cycle ◽  
Reading Frame

ABSTRACT We used a photoactivatable, lipophilic reagent, 3′-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies ofChlamydia trachomatis LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.

scholarly journals Immunoisolaton of the Yeast Golgi Subcompartments and Characterization of a Novel Membrane Protein, Svp26, Discovered in the Sed5-Containing Compartments

2005 ◽  
Vol 25 (17) ◽  
pp. 7696-7710 ◽  
Author(s):  
Hironori Inadome ◽  
Yoichi Noda ◽  
Hiroyuki Adachi ◽  
Koji Yoda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Membrane Protein ◽  
Considerable Portion ◽  
Marker Proteins ◽  
Evolutionarily Conserved ◽  
Golgi Compartment

ABSTRACT The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

scholarly journals Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

2015 ◽  
Vol 396 (8) ◽  
pp. 903-915 ◽  
Author(s):  
Nazhat Shirzad-Wasei ◽  
Jenny van Oostrum ◽  
Petra H.M. Bovee-Geurts ◽  
Lisanne J.A. Kusters ◽  
Giel J.C.G.M. Bosman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Partial Purification ◽  
Functional Studies ◽  
Host Membrane ◽  
Lipid Nanodiscs ◽  
Membrane Scaffold ◽  
Rapid Transfer

Abstract Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

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 Mass spectrometry of membrane protein complexes

2019 ◽  
Vol 400 (7) ◽  
pp. 813-829 ◽  
Author(s):  
Julian Bender ◽  
Carla Schmidt
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Membrane Protein Complexes ◽  
Hydrophobic Nature ◽  
Alternative Approaches ◽  
Lipid Environment ◽  
And Function

Abstract Membrane proteins are key players in the cell. Due to their hydrophobic nature they require solubilising agents such as detergents or membrane mimetics during purification and, consequently, are challenging targets in structural biology. In addition, their natural lipid environment is crucial for their structure and function further hampering their analysis. Alternative approaches are therefore required when the analysis by conventional techniques proves difficult. In this review, we highlight the broad application of mass spectrometry (MS) for the characterisation of membrane proteins and their interactions with lipids. We show that MS unambiguously identifies the protein and lipid components of membrane protein complexes, unravels their three-dimensional arrangements and further provides clues of protein-lipid interactions.

scholarly journals Structural predictions of the functions of membrane proteins from HDX-MS

2020 ◽  
Vol 48 (3) ◽  
pp. 971-979
Author(s):  
Andy M. Lau ◽  
Ruyu Jia ◽  
Richard T. Bradshaw ◽  
Argyris Politis
Keyword(s):  
Experimental Data ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Short Review ◽  
Fundamental Knowledge ◽  
Significant Potential ◽  
Structure And Dynamics ◽  
Recent Advances ◽  
Lipid Environment ◽  
Hdx Ms

HDX-MS has emerged as a powerful tool to interrogate the structure and dynamics of proteins and their complexes. Recent advances in the methodology and instrumentation have enabled the application of HDX-MS to membrane proteins. Such targets are challenging to investigate with conventional strategies. Developing new tools are therefore pertinent for improving our fundamental knowledge of how membrane proteins function in the cell. Importantly, investigating this central class of biomolecules within their native lipid environment remains a challenge but also a key goal ahead. In this short review, we outline recent progresses in dissecting the conformational mechanisms of membrane proteins using HDX-MS. We further describe how the use of computational strategies can aid the interpretation of experimental data and enable visualisation of otherwise intractable membrane protein states. This unique integration of experiments with computations holds significant potential for future applications.

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