scholarly journals DNA-Mediated Stack Formation of Nanodiscs

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1647
Author(s):  
Madhumalar Subramanian ◽  
Charlotte Kielar ◽  
Satoru Tsushima ◽  
Karim Fahmy ◽  
Jana Oertel
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Crystallization ◽  
Fluorescent Labeling ◽  
Size Exclusion ◽  
Scaffolding Protein ◽  
Limiting Factor ◽  
Protein Ratio ◽  
Flexible Spacer ◽  
Thiol Reactivity

Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.

scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

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 An automated platform for structural analysis of membrane proteins through serial crystallography

2021 ◽  
Author(s):  
Robert D Healey ◽  
Shibom Basu ◽  
Anne-Sophie Humm ◽  
Cedric Leyrat ◽  
Xiaojing Cong ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
High Throughput ◽  
Drug Targets ◽  
Protein Crystallization ◽  
Integral Membrane Protein ◽  
Structural Level ◽  
Ligand Discovery ◽  
Serial Crystallography ◽  
Automated Platform

Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze at a structural level. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins due to lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperature and in a single support. We apply this approach to two human integral membrane proteins, which allowed us to capture different conformational states of intramembrane enzyme-product complexes and analyze the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting and serial crystallography enabling screening of small molecule libraries with membrane protein crystals grown in meso. This approach brings badly needed automation for this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins.

scholarly journals Be Cautious with Crystal Structures of Membrane Proteins or Complexes Prepared in Detergents

Crystals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 86 ◽  
Author(s):  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Crystal Structures ◽  
Structure Determination ◽  
Protein Crystallization ◽  
Energy Coupling ◽  
Careful Examination ◽  
Cubic Phase ◽  
Native Cell ◽  
Membrane Protein Crystallization

Membrane proteins are an important class of macromolecules found in all living organisms and many of them serve as important drug targets. In order to understand their biological and biochemical functions and to exploit them for structure-based drug design, high-resolution and accurate structures of membrane proteins are needed, but are still rarely available, e.g., predominantly from X-ray crystallography, and more recently from single particle cryo-EM — an increasingly powerful tool for membrane protein structure determination. However, while protein-lipid interactions play crucial roles for the structural and functional integrity of membrane proteins, for historical reasons and due to technological limitations, until recently, the primary method for membrane protein crystallization has relied on detergents. Bicelle and lipid cubic phase (LCP) methods have also been used for membrane protein crystallization, but the first step requires detergent extraction of the protein from its native cell membrane. The resulting, crystal structures have been occasionally questioned, but such concerns were generally dismissed as accidents or ignored. However, even a hint of controversy indicates that methodological drawbacks in such structural research may exist. In the absence of caution, structures determined using these methods are often assumed to be correct, which has led to surprising hypotheses for their mechanisms of action. In this communication, several examples of structural studies on membrane proteins or complexes will be discussed: Resistance-Nodulation-Division (RND) family transporters, microbial rhodopsins, Tryptophan-rich Sensory Proteins (TSPO), and Energy-Coupling Factor (ECF) type ABC transporters. These analyses should focus the attention of membrane protein structural biologists on the potential problems in structure determination relying on detergent-based methods. Furthermore, careful examination of membrane proteins in their native cell environments by biochemical and biophysical techniques is warranted, and completely detergent-free systems for membrane protein research are crucially needed.

scholarly journals Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein

1995 ◽  
Vol 312 (2) ◽  
pp. 543-548 ◽  
Author(s):  
P E Golstein ◽  
A Sener ◽  
R Beauwens
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Sodium Cholate ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Iodide Uptake ◽  
Protein Insertion ◽  
Set Up

A sensitive assay is developed to assess the existence of an iodide channel in a fraction of solubilized membrane proteins. This step is critical when considering various procedures for purification of this channel. Sodium cholate is used as a detergent as it does not denature the iodide channel. A simple and rapid method involving gel-filtration chromatography is used simultaneously to remove the detergent and to adjust the buffer composition, before protein insertion into liposomes. The presence of an iodide channel is investigated by measuring the iodide conductance of these proteoliposomes at 4 degrees C. An outward iodide gradient is set up across the proteoliposomal membrane by anion-exchange chromatography, allowing uptake of radiolabelled iodide. This uptake is conductive as it is abolished by valinomycin in the presence of potassium. It is specifically mediated by a thyroid plasma-membrane protein inserted into liposomes, as its denaturation before insertion totally abolished uptake. It was observed only within a well-defined fraction of thyroid membrane proteins collected by size-exclusion chromatography (molecular mass between 100 and 200 kDa). Furthermore, it was not observed with other membrane proteins such as ileal brush-border-membrane proteins or bacteriorhodopsin. Like many anion channels, this conductance was also inhibited by N-phenylanthranilic acid. Optimization of the assay is described, validating the measurement of conductive iodide uptake at 30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of protein to 90 micrograms of lipid, with an outward iodide gradient (KI 15 mM inside and 1 microM outside). This assay provides a test of the biological activity of the iodide channel at each step of the purification; it can be applied to any anionic channel.

Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins

2003 ◽  
Vol 36 (6) ◽  
pp. 1372-1377 ◽  
Author(s):  
Vadim Cherezov ◽  
Martin Caffrey
Keyword(s):  
Optical Properties ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Low Cost ◽  
Protein Crystallization ◽  
Batch Crystallization ◽  
Membrane Protein Crystallization ◽  
Crystallization Trial

A simple convenient and low-cost glass-based plate for high-throughput screening of membrane protein crystallization is described. The plates are robust and reduce dramatically the amount of protein and precipitant solution used per crystallization trial, while offering excellent optical properties for the detection of micro-crystals and crystals of colorless proteins. The plates were developed primarily for crystallization of membrane proteins in lipidic mesophases. They can also be used in batch crystallization of soluble and membrane proteins.

scholarly journals Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Kanchan Gupta ◽  
Gilman ES Toombes ◽  
Kenton J Swartz
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Conformational Changes ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Bioorthogonal Chemistry ◽  
Kv Channel ◽  
Cysteine Mutagenesis ◽  
Voltage Dependent ◽  
Methionine Residues

The functional mechanisms of membrane proteins are extensively investigated with cysteine mutagenesis. To complement cysteine-based approaches, we engineered a membrane protein with thiol-independent crosslinkable groups using azidohomoalanine (AHA), a non-canonical methionine analogue containing an azide group that can selectively react with cycloalkynes through a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We demonstrate that AHA can be readily incorporated into the Shaker Kv channel in place of methionine residues and modified with azide-reactive alkyne probes in Xenopus oocytes. Using voltage-clamp fluorometry, we show that AHA incorporation permits site-specific fluorescent labeling to track voltage-dependent conformational changes similar to cysteine-based methods. By combining AHA incorporation and cysteine mutagenesis in an orthogonal manner, we were able to site-specifically label the Shaker Kv channel with two different fluorophores simultaneously. Our results identify a facile and straightforward approach for chemical modification of membrane proteins with bioorthogonal chemistry to explore their structure-function relationships in live cells.

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

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Fei Jin ◽  
Cheng Shen ◽  
Yao Wang ◽  
Mengqi Wang ◽  
Minxuan Sun ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Size Exclusion Chromatography ◽  
Fluorescence Detection ◽  
False Negative ◽  
Membrane Topology ◽  
Size Exclusion ◽  
Expression Screening ◽  
Cell Extracts ◽  
Exclusion Chromatography

AbstractGFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. 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 for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.

An efficient nanolitre-volume multi-channel device for highly viscous materials used in membrane protein crystallization

2013 ◽  
Vol 46 (3) ◽  
pp. 829-831
Author(s):  
Jinghui Luo ◽  
Raphaël Zwier ◽  
Jan Pieter Abrahams
Keyword(s):  
Lower Bound ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Crystal Structures ◽  
Protein Crystallization ◽  
Cubic Phase ◽  
Operation Speed ◽  
Membrane Protein Crystallization ◽  
Lipidic Cubic Phase ◽  
Materials Used

The crystal structures of various important membrane proteins could not have been solved without lipidic cubic phase (LCP) crystallization, and yet, compared to traditionalin surfocrystallization, LCP crystallization is not widely used because its extreme viscosity makes the cubic phase difficult to handle. Robots that can dispense LCPs are very specialized and therefore very expensive. Here, an accurate multi-channel device is described. It dispenses LCPs onto glass plates down to volumes of 20 nl accuracy and has an accuracy of 10% when dispensing 200 nl – the lower bound of LCP volumes dispensed for crystallization trials. Because of its multi-channel tips, operation speed goes up by a factor of four compared to simpler devices. It can be operated by hand, but its design also allows it to be built into a basic dispensing robot. Thus, the device lowers the threshold for LCP crystallization of membrane proteins/peptides.

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