Novel systematic detergent screening method for membrane proteins solubilization

Accepted Manuscript Novel systematic detergent screening method for membrane proteins solubilization Elodie Desuzinges Mandon, Morgane Agez, Rebecca Pellegrin, Sébastien Igonet, Anass Jawhari PII:

S0003-2697(16)30390-6

DOI:

10.1016/j.ab.2016.11.008

Reference:

YABIO 12554

To appear in:

Analytical Biochemistry

Received Date: 26 September 2016 Revised Date:

20 October 2016

Accepted Date: 10 November 2016

Please cite this article as: E.D. Mandon, M. Agez, R. Pellegrin, S. Igonet, A. Jawhari, Novel systematic detergent screening method for membrane proteins solubilization, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.11.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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TITLE: Novel systematic detergent screening method for membrane proteins solubilization AUTHORS: Elodie DESUZINGES MANDON1*, Morgane AGEZ1*, Rebecca PELLEGRIN1*, ¶

1

CALIXAR, 60 avenue Rockefeller, 69008 Lyon, France;

*

Contributed equally to this work Corresponding author: [email protected]

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¶

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Sébastien IGONET1 & Anass JAWHARI1

Running title: New solubilization screening for membrane proteins

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Keywords: Extraction, solubilization, detergent, calixarene, membrane protein, biotinylated lipids, membranes, GPCR, transporter.

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ABSTRACT Membrane proteins play crucial role in many cellular processes including cell adhesion, cell-cell communication, signal transduction and transport. To better understand the molecular basis of such central biological machines and in order to

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specifically study their biological and medical role, it is necessary to extract them from their membrane environment. To do so, it is challenging to find the best solubilization condition. Here we describe, a systematic screening method called BMSS (Biotinylated Membranes Solubilization & Separation) that allow screening 96 conditions at once. Streptavidine magnetic beads are used to separate solubilized

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proteins from remaining biotinylated membranes after solubilization. Relative quantification of dot blots help to select the best conditions to be confirmed by

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classical ultra-centrifugation and western blot. Classical detergents with different physical-chemical characteristics, novel calixarene based detergents and combination of both, were used for soubilization trials to obtain broad spectrum of conditions. Here, we show the application of BMSS to discover solubilization conditions of a GPCR target (MP-A) and a transporter (MP-B). The selected conditions allowed the solubilization and purification of non-aggregated and homogenous native membrane

throughput

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proteins A and B. Taken together, BMSS represent a rapid, reproducible and high assessment

of

solubilization

toward

biochemical/

functional

characterization, biophysical screening and structural investigations of membrane

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proteins of high biological and medical relevance.

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INTRODUCTION Integral membrane proteins play crucial role in many cellular processes including cell adhesion, cell-cell communication, immunity, signal transduction and transport. Biological membranes are made of diverse type of lipids and proteins [1,2]. This makes them of high interest for fundamental research and therapeutic purposes since

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60% of therapeutic targets are membrane proteins [3]. Genes coding for integral membrane proteins such as transporters, G protein coupled receptors, ion channels or enzymes represent 20-30% of protein coding genes [4]. Despite the growing interest, studying membrane proteins out of their natural lipid environment represents a major

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challenge. Indeed, integral membrane proteins are believed to be more challenging than soluble proteins due to their hydrophobic nature and stable form at the membrane

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[5,6,7]. Structural studies and biophysical screening of integral membrane proteins (MP) require often a recombinant expression, MP solubilization from membrane environment and purification. Several attempts have been made to facilitate the extraction of membrane proteins from biological membranes for subsequent structural and functional characterizations [8,9,10]. This is still a very limiting step that is exceptionally time and resource consuming without much of success. One of the

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reasons is certainly the limiting number of solubilization trials and the poor diversity of detergents/ surfactants used for MP extraction and purification. In fact, 252 unique integral membrane protein structures have been solved by X-ray crystallography up to 2015 [11]. Only 5 detergents (DDM, DM, NG, OG and LDAO) have yielded the

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majority (more 60%) of alpha helical membrane protein structures [12,13]. This is limited given the biochemical diversity of MP families, with selection based primarily

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on previous successes of other MPs. The prediction of the suitable detergent for each protein or class of protein is still not realistic and remains out of reach. In addition to that, no single detergent or family of detergent can be considered to be the best detergent suitable for solubilization of all MPs, as the molecular principles of solubilization remain unclear. For these reasons, a systematic detergents screening assay including a wide range of detergent with different physical-chemistry features is required to tackle solubilization as one of the major obstacles for MP production and characterization. Similarly, other trials to facilitate the production and crystallization of membrane proteins have been described [5,14,15,16,17,18,19,20,21]. In addition to detergent solubility studies [7,9,22], including size exclusion chromatography or

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FSEC [19,23,24], ultracentrifugation dispersity sedimentation assay [25] and differential filtration assay [26].

Here we describe a method called BMSS

(Biotinylated Membranes Solubilization & Separation) that uses streptavidin binding of solubilized biotinylated membranes. Previously, the ability of streptavidin to bind tightly biotinylated lipid layers has been applied for Surface Plasmon resonance to

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study protein/ ligands interactions [27,28] and to study Giant Unilamelar Vesicles in the context of Apolipoprotein biology [29]. Here we describe, a systematic screening method BMSS (Biotinylated Membranes Solubilization & Separation) that allow screening 96 conditions at once. Streptavidine magnetic beads allow separation of

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solubilized proteins from remaining biotinylated membranes after solubilization. This assay is based on the assumption that non-solubilized membranes are orders of magnitude more biotinylated than solubilized protein/ lipid/ detergent complexes and

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therefore can be easily separated from solubilized proteins. Supernatant was then analyzed by dot blot to find the best solubilization conditions. The result was further confirmed by classical ultra-centrifugation followed by western blot. Classical detergents with different physical-chemical characteristics, novel calixarene based detergents [7,30,31] and combination of both, were used for solubilization trials to

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reach a broad spectrum of conditions. We have applied this assay to several membrane proteins of high medical relevance. Results are shown for 2 recombinantly expressed proteins MP-A (belonging to GPCR family) and MP-B (belonging to transporter family) for which discovered solubilization conditions could led to the

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purification of non-aggregated and homogenous proteins. Taken together, BMSS represent a rapid, reproducible and high throughput assessment of solubilization, as

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key step toward biochemical/ functional characterization, biophysical screening and structural investigations of membrane proteins of high biological and medical importance.

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RESULTS Ultracentrifugation is the common way to separate solubilized membrane protein from insoluble fraction. Although fast and easy to implement with few detergents, this method can become laborious and time consuming when it comes to screen high number of detergents. In order to screen large number of detergent conditions, we

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initially thought about applying filtration steps to separate solubilized protein for membranes. This idea cannot apply to all proteins since each protein has different molecular weight/ size and filter cut-off would need to be adjusted each time. We then thought about biotinylating the membranes in order to separate

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them from solubilized material. Biotinylation of biological membranes is not very efficient and would require specific treatment and optimization n addition

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to the fact that it may results on biotinylating proteins as well which make the separation very complicated. This is why we thought about integrating biotinylated lipids into biological membrane not for fusion purposes but only to be able to separate membranes from solubilized proteins. Indeed, the idea is to apply detergent above the CMC to biotinylated membrane/ protein complexes and use magnetic beads to strip the highly biotinylated lipid complexes out of the

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solution, leaving the solubilized proteins in solution to be analyzed. Indeed, here we report a novel alternative screening method in a 96-well microplate format, called BMSS (Biotinylated Membranes Solubilization & Separation). This assay is based on the assumption that non-solubilized membranes are orders of magnitude more

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biotinylated than solubilized protein/ lipid/ detergent complexes. Figure 1 illustrates the overall method and key steps. Typically, the first step consists in inserting

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biotinylated liposomes into biological membranes (plasma membranes, internal membranes, exosomes, endosomes, liposomes..). To do so, the biotinylated lipids are incubated over-night at 4°C under gentle agitation with the biological membranes with a 1 to 1 (m/m) ratio. Non-inserted biotinylated lipids were separated by sucrose gradient (see Mat & methods and Figure 1). The 1/1 ratio was a default value that we decided to apply at first trial. Different incubation times were applied for the stretpavidin magnetic beads binding to be able to have a clear difference between SDS- solubilized sample and non- solubilized membranes (Figure 2B). 15 minutes incubation was enough to have an efficient separation (Figure 2B). More than 1 hour incubation can led to binding

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solubilized protein as they contain mixed micelles of detergent/ lipids. What we aimed for is to have a decent amount of membranes that are biotinylated and not to reach 100%. The yield is generally around 50%. High biotinylation yield can be problematic since protein/ biotinylated lipids complexes can be overrepresented and therefore such complex will be binding to the streptavidin resin

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making the interpretation of the solubilization result difficult. Solubilization screening will be using the same membrane preparation for all detergents conditions to avoid variability. What is important is to be able to see differences between “good” and “bad” solubilization conditions. Membrane protein

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solubilization is then performed on biotinylated membranes (Figure 1, step 3) using classical detergents, novel calixarenes (see table S1) or a combination of both (see table S2-8). After solubilization, the incubation with streptavidin magnetic beads

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allows the pull-down of insoluble biotinylated membranes (step 4). The unbound solubilized fraction can be then easily collected and analyzed by Dot-Blot. Since one of the key steps of this method is to biotinylate biological membranes, we needed to ensure that biotinylated lipids were successfully inserted into biological membranes. As shown in Figure 2A, Dot Blot analyses using streptavidin coupled antibody was

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performed and could confirm biotinylation of the membranes. As positive control, biotinylated vesicles/ liposomes show specific signal in the same assay. The presence of the membrane protein of interest (MP-A or MP-B) was observed in both biotinylated and non-biotinylated biological membranes. Biotinylated lipid insertion

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was therefore validated. We then applied BMSS to study solubilization of two very different membrane proteins MP-A and MP-B belonging to two families, GPCR and

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Transporter, respectively and expressed in two cell membrane types (insect cells and bacteria, respectively). Biotinylated membranes were solubilized by addition of SDS and submitted either to Streptavidin-magnetic beads separation (Figure 2.B) or ultracentrifugation (Figure 2.C) to separate insoluble from soluble material. Using both methods, the protein was detected in the supernatant fraction only due to SDS solubilization. This demonstrates that MP-A and MP-B were present at the biotinylated plasma membrane and not in the soluble fraction. In order to verify the reproducibility of the method, and to study different detergents effects, we decided to focus on one membrane protein target and apply solubilization trials using classical detergents (DDM, OG, C12E8, CHAPS, LDAO, FC12 and Sarkozyl), or novel

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detergents

[7,30,31]

with

different

physical-chemical

features

(CALX153ACE, CALX163ACE, CALX173ACE, CALX173GLUK, CALX183ACE, CALX193ACE, CALX1103ACE, CALX1113ACE and CALX1123ACE) or a combination of both (Figure 3 and table S1). No detergent served as negative control. Whereas, SDS served only as solubilization indicator and not a good positive control

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since its denaturating/ solubilizing characteristics are protein and membrane dependent. BMSS is not an absolute assessment of solubilization rate but a relative estimate of better solubilization conditions that show the best dot signal intensities. The separation between solubilized and non-solubilized protein (Figure 1, step 4) was

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done twice and dot blots were performed twice for each separation experiment. Figure 3 showed very similar dot blot profile for the duplicate of duplicate. Table S2 indicated the result of the quantification of the dot blots with low error bars,

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demonstrating the sensitivity and consistency of the data. Regarding the use of classical detergents, LDAO and Sarkozyl gives good results with a very good solubilization rate for the later (table S2). This is not a surprise giving that Sarkozyl is very well known to be a harsh detergent. Interestingly, several calixarene-based detergents could also solubilize MP-A. This is consistent with the good solubilization

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characteristics of such class of detergents [7,30,31]. The combination of each Calixarene based detergent CALX173ACE or CALX1103ACE with eight classical detergents show that solubilization rate may significantly change from low to high (for example DDM and DDM/CALX1103ACE) or the opposite (for example

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CALX173ACE and CALX173ACE/CHAPS), illustrating therefore the value of combining compounds (table S2). Since the data was reproducible and consistent with

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what was expected from MP solubilization trials, we decided to increase the number of solubilization conditions and to extend the study to other classical detergents, novel calixarenes and new resulting combinations. Therefore a screen of 96-solubilization set-up was applied to MP-A as well as MP-B (Figure 4, table S3 and S4). Since MP-A and MP-B are different targets that belong to 2 distinct classes of membrane proteins, we expect to have different solubilization profiles. This is exactly what Figure 4 shows. In fact, while MP-A show more solubilization conditions (see table S3), MP-B show a typical profile of a very difficult to extract MP (see table S4). Indeed, only two conditions using Calixarene detergent CALX173ACE in combination with nonionic molecules such as DDM and Triton allowed good solubilization (above 70%). These differences between MP-A and MP-B solubilization profiles demonstrate the 7

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specificity and the sensitivity of the method and the possibility to apply it for both easy and difficult to extract membrane proteins. In order to evaluate correlation between classical ultracentrifugation and BMSS described here, we have selected representative of good (more than 50% solubilization yield, Figure 5 represented in red) and bad (less than 50% solubilization yield, Figure 5 represented in blue)

submitted

the

corresponding

membranes

to

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solubilization conditions for MP-A and MP-B (17 and 10, respectively) and we have solubilization

followed

by

ultracentriguation. The results shown in Figure 5, corresponds to SDS-PAGE followed by western blot using antibodies against MP-A or MP-B. The quantification

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of MP-A and MP-B bands in the soluble and insoluble fractions was used to evaluate the solubilization yield (table S5 and S6). Since most of the observed signal corresponds to monomeric forms of the proteins, only bands of the monomers were

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used for relative quantifications. Tables S5 and S6 show the results of such solubilization rate evaluation for MP-A and MP-B, respectively. For MP-A, as indicated in Figure 5A and Table S5, 5 of the best-solubilized conditions obtained using BMSS were confirmed by classical ultracentrifugation and western blot (CALX183ACE, LMNG/CALX173ACE, LDAO/CALX173ACE, Sarkozyl and

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Sarkozyl/CALX173ACE). BMSS show also other good solubilization conditions such as CALX173ACE, CALX193ACE and CALX173ACE/DDM. For MP-B, as shown in Figure 5B and Table S6, out of 3 good solubilization conditions obtained using BMSS described here, 3 were also confirmed by classical ultracentrifugation and

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western blot (CALX153ACE, CALX173ACE and CALX173/Triton, respectively). Even if the two methods are extremely different from the experimental setting point

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of view, the obtained results show strong similarities. In order to evaluate potential variability between different membranes batches, we decided to apply BMSS to membranes resulting from two independent Sf9 expression of MP-A. Table S7 shows high similarities for all solubilization conditions, demonstrating the reproducibility and robustness of BMSS. Since finding relevant solubilizing conditions represent the first step toward MP preparation and characterization, we have chosen one good solubilization condition per protein for which we could establish affinity purification. Figure 6A show specific binding and elution of MP-A and MP-B on His-tag affinity chromatography. MP-A was less abundant than MP-B therefore western blot and coomassie staining were performed, respectively. The solubilization conditions established by BMSS allowed purifying the proteins that behaves as homogenous and 8

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non aggregated in Native PAGE (Figure 6B). This was further confirmed by negative staining electron microscopy (Figure 6C) demonstrating that BMSS could be used to identify solubilization conditions that result on homogenous and non-aggregated protein.

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purified

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DISCUSSION The extraction of integral membrane proteins from their natural lipid environment requires specific balance through the choice of surfactants with capabilities to disrupt the tight interactions between proteins and lipids without disrupting the protein structure and function. Because of the wide range of biochemical diversity on

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membrane protein families, classes and types, it is unlikely that a single detergent or group of detergent will be able to balance between efficient solubilization and conservation of the structural and functional integrities. Extraction efficiency can be dramatically variable with different detergents for the same protein. Despite

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significant effort, the molecular mechanisms that control membrane protein solubilization and the interaction between protein and lipids have not been fully

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understood [32,33,34,35]. For these reasons, it is of high interest to develop methods to systematically screen detergents and/ or surfactants with different physicalchemical properties in order to fine-tune specific and unique solubilization condition for each membrane protein from each biological membrane with its specific lipid composition

(plasma membrane,

internal

membrane,

exosome,

endosomes,

liposomes..). Recent report of detergent screening for solubilization and purification

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of membrane proteins was described [10]. This work combined membrane solubilization and affinity chromatography and therefore does not allow the use of large number of detergents, to quality control each step separately and do not apply to untagged proteins. Here we report a novel solubilizing screening method BMSS in a

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96-well microplate format. This consist on using biotinylated lipids that we specifically integrate into biological membranes, then using streptavidine magnetic

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beads, non-solubilized membranes are pulled-down. This assay is based on the idea that non-solubilized membranes are orders of magnitude more biotinylated than solubilized protein/ lipid/ detergent complexes and can easily be separated from solubilized protein. We cannot exclude that biotinylated lipid insertion into biological membranes may have some effect on the protein function. However, biotinylation is not used to purify protein, but only for the solubilization screening that help to identify the first solubilization hits that are then confirmed by western blot and the resulting purified protein is then tested for its behaviour in solution/ aggregation as shown here for the 2 examples (GPCR and transporter). Other proteins such as ion channels or GPCR for which such screening has been applied show good

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functionality. Indeed, after solubilization and purification of native human GPCR (adenosine receptor) could show specific radiobinding to an antagonist compound and show similar binding affinities to what was previously established (Igonet S et al., unpublished). Similarly matrix 2 ion channel from the influenza virus was solubilized from MDCK infected cells and after purification of

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tetramers could show specific proton selective ion channel activity that was specifically inhibited by addition of specific inhibitor the amantadine (Mandon. E et al., Protein Expression & Purification, revision).

This method represents an alternative to ultracentrifugation or filtration. BMSS is high

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throughput (HT) compatible and can be integrated into the workflow of HT membrane protein production for small molecules biophysical screening, antibody

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display and structural genomic. BMSS can be applied to all type of tagged proteins as well as to untagged protein resulting from endogenous expression for instance. BMSS could also be used to screen different pH, salt concentrations, and addition of specific ligands. This method has been already used in the laboratory to allow production of well-folded and functional protein for drug discovery purposes. MP-A (GPCR) and MP-B (transporter) described here belong to 2 families of membrane proteins,

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expressed in two expressing systems with very different expression yield. BMSS was also applied to the same protein (MP-A) expressed in two expression systems (insect cells and yeast, table S8). Interestingly, BMSS could show very similar tendencies for solubilization with good conditions being CALX183ACE, DDM/CALX173ACE and

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FC14/CALX193ACE for example. In general, MP-A from yeast was less solubilized in comparison to Sf9. This is true mainly for single solubilization trials and do not

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apply to double solubilization where good solubilization conditions were identified. BMSS may be used in the future to delineate the determinants of solubilization when applied to the same protein expressed in different expression systems and the same system expressing different proteins belonging to the same or very different MP family. BMSS combined with molecular simulation would provide response elements on the contribution of protein abundance/ expression, protein fold/ structure, as well as lipid composition on solubilization efficacy, which would help identify good solubilization conditions but maintain the structural integrity as described here for MP-A and MP-B. In fact, these preparations could lead to non-aggregated and relatively homogenous protein (Figure 6). The use of calixarenes based detergents

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alone and/ or in combination with classical detergents as this is the case for MP-A and MP-B, have been recently reported to help stabilize well-folded and functional membrane proteins [7,30,31]. Regarding the reproducibility of the method, the Dot Blots were done on duplicates of duplicates, showing relatively low quantification errors considering the variability of detection methods with antibodies such as

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western blot. The same reproducibility was observed when two batches of Sf9 expressing MP-A were used (table S7). Despite the expression yield and difference of expression system for MP-A (insect cells, yeast) and MP-B (E. coli), BMSS could be applied successfully, demonstrating the applicability of BMSS for different protein

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expression settings. We have noticed that for extremely low expressed proteins (less than 50 microgram of protein per liter of culture), the quantification errors can increase due to low signal to noise ratio (data not shown). In addition to the

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advantages of BMSS in term of screening large number of detergents/ surfactants and the associated success rate for the production of well-folded and functional membrane proteins, it is important to stress that the screening of 96 conditions is rapid and requires very low amount of detergent. This is very convenient since novel detergents are not always affordable and large screening is time consuming. BMSS can readily be

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extended to the screening of GFP fused proteins in order to measure direct fluorescence and with the limitation of working with a fusion protein and not the native sequence. In the near future BMSS will be coupled to affinity purification, radioligand binding (for GPCR) or current voltage measurement (on ion channels)

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and dynamic light scattering.

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MATERIALS & METHODS

CELL LYSIS AND MEMBRANE FRACTIONATION

The full–length human GPCR MP-A (GenBank Accession: AY136747) was cloned

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into pOET1 transfer plasmid in frame with N-terminal hemagglutinin signal sequence, Strep-tag II and 8xHis tag. Baculovirus was produced according to the manufacturer’s protocol (flashback ULTRA™ system, Oxford Expression Technologies). For protein expression, Sf9 insect cells were infected with baculovirus as described [36] at a

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density of 1.5x106 cells ml−1, using 20 µl of baculovirus stock per 1.0x106 cells, and grown at 28 °C for 64 hours in an orbital shaker. After 64 hours, cell pellets were collected, washed in 50 mM HEPES buffer pH 7.4 with 200mM NaCl, then stored at -

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80°C until use. Yeast expression was performed as described [37].

The full–length transporter MP-B (GenBank Accession: KIX83253.1) was expressed as described [31], in E. Coli Strain CD41(DE3) was induced with 0.7mM Isopropyl β-D-1- thiogalactopyranoside (IPTG) for 3h at 25°C after cells reached an OD600 of 0.5. Cells pellets were then collected, flash-frozen and stored at -80°C until use. MP-

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A frozen pellets were thawed, resuspended and lysed by mechanical cell lysis in PBS complemented with Protease Inhibitor Cocktail 1X (PIC). Mechanical cell lysis was performed on ice using a Bead Beater homogenizer with 0.1mm diameter glass beads. Membrane fractionation was then carried out at 4°C by sequential centrifugations

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(1000g for 5min, 15000g for 30min, and 100000g for 45min). Plasma membranes (pellet 100000g) were resuspended in Phosphate buffered saline (PBS) 1X, PIC 1X,

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glycerol 20%, quantified using the Pierce Micro BCA Protein Assay Kit (Thermo Scientific), flash-frozen and stored at -80°C until use. MP-B Frozen cell pellets were thawed, resuspended and lysed using a French press (16000psi) in Tris 50mM pH8, MgCl2 5mM, Dithiothreitol

(DTT) 1mM complemented with Protease Inhibitor

Cocktail 1X (PIC). Membrane fractionation was then carried out at 4°C by sequential centrifugations (4000g for 10min, and 100000g for 45min). Plasma membrane (pellet 100000g) were resuspended in Tris 20mM pH 8, sucrose 300mM, EDTA 1mM PIC 1X, glycerol 10%, quantified using the Pierce Micro BCA Protein Assay Kit (Thermo Scientific), flash-frozen and stored at -80°C until use.

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SOLUBILIZATION OF NON-BIOTINYLATED PLASMA MEMBRANE All chemicals for the detergent panel were obtained from CALIXAR, Anatrace, Roth, Euromedex, Affimetrix and Sigma-Aldrich, as indicated in Table S1. Working stock solutions (2X) were made in ultrapure water, dispensed into 96-well plates, sealed and frozen at –20 °C until needed. For solubilization trials, 100µl of Plasma membrane

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proteins MP-A and MP-B were incubated under a final volume of 50 µl for 2h at 4°C in a final concentration of 1mg/ml in a PBS buffer containing 1X PIC and 10-fold the Critical Micelle Concentration (CMC) of detergent. Removal of insoluble material was performed by ultracentrifugation 1h, 100000g at 4°C. Total extract, pellet and

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soluble fractions were analyzed by SDS-PAGE and Western-blot as described below.

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SDS-PAGE AND WESTERN-BLOT

Samples were mixed with a 5x loading dye then loaded on a 4–15% MiniPROTEAN® TGX Stain-Free™ Gel, using Tris Glycine (TG)- Sodium Dodecyl Sulfate (SDS) running buffer. Proteins were subsequently transferred to PVDF membranes, blocked with Roti block buffer (ROTI, Carl Roth KG) and probed with either a mouse anti-MP-A/B antibody followed by an anti-mouse IgG-HRP

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(horseradish peroxidase)-conjugated antibody or coomassie staining for MP-B protein. Final detection for MP-A was made using the ChemiDoc MP (Bio-Rad). The signal of the protein observed in western blot in the P, S and T were integrated using ImagLab software (biorad) to determine the % of solubilization that is equal the

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Intensity of S/ (Intensity P + Intensity of S). The background was calculated for each

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spot from the median value of the surrounding baseline.

BMSS (BIOTINYLATED MEMBRANE SOLUBILIZATION AND SEPARATION) PREPARATION OF BIOTIN-DHPE LIPOSOME Biotin-DHPE liposomes were obtained by resuspension in chloroform, slow evaporation of the solvent using a rotary evaporation system (Hei-VAP, Heidolph), followed by rehydration in PBS buffer at a concentration of 10mg/ml with vortexing and sonication in a bath sonicator (2510E-MT, Branson) for 30min to homogenize liposome particles.

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PREPARATION OF BIOTINYLATED PLASMA MEMBRANE Insertion of biotin-DHPE in plasma membrane was obtained by o/n incubation at 4°C under agitation in a 1 to 1 (m/m) ratio. Non-inserted Biotin DHPE (N(Biotino yl)-1,2-Dihexadecano yl-sn-Gl ycero-3-Phosphoethanolamine, Trieth ylammonium Salt) liposomes were separated from plasma membrane by

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sucrose density gradient: the Biotin-DHPE/plasma membrane mixture was overlaid on a 5ml centrifuge tube and sequentially layered with lower concentration of sucrose in PBS (1.2ml of 60% sucrose, 1.2ml of 25% sucrose and 1.2ml of 10% sucrose). After centrifugation at 200000g (Rotor S52ST-0330, Thermo Scientific) for 1h at

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4°C, biotinylated plasma membrane was collected, washed with 3 volume of PBS and centrifuged at 35000g for 30min at 4°C. Biotinylated plasma membrane pellet was resuspended in PBS-PIC 20% glycerol, quantified using the Pierce Micro BCA

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Protein Assay Kit (Thermo Scientific), then flash-frozen in liquid nitrogen until use.

MICROPLATE SOLUBILIZATION SCREEN OF BIOTINYLATED PLASMA MEMBRANE

For micro-plate solubilization, typically 25µl of biotinylated plasma membrane at 2mg/ml was mixed with 25µl of 20CMC of detergent stock solution. Removal of

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insoluble material was performed by Streptavidin/biotin-affinity chromatography. Total extracts were incubated on 20µl of pre-equilibrated Mag Strep resin slurry (1µl resin, Mag Strep type 3 XT, IBA) at 4°C for 15min under agitation. The microplate was then placed on a magnetic separator and the supernatant, corresponding to the

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soluble fraction, was collected and analyzed by Dot-Blot as described below in duplicate of duplicates.

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DOT-BLOT

50µl of solubilized fractions were loaded on a nitrocellulose membrane (GE Healthcare Life Sciences Amersham) using the Bio-Dot® 96 wells Microfiltration Apparatus (BIO-RAD). Depending of the intensity of the signal, solubilized fraction may be diluted prior to loading on the nitrocellulose membrane. Nitrocellulose membranes were then blocked with Roti block buffer (ROTI, Carl Roth KG) and probed with either a mouse anti-MP-A antibody followed by an anti-mouse IgG-HRP (horseradish peroxidase)-conjugated antibody for the detection of MP-A or by an antiHis-HRP conjugated antibody for the detection of MP-B, using therefore

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chemiluminescence chemistry for both detections.

Final detection was made using

the ChemiDoc MP (Bio-Rad). Integrated spot intensities were measured with the ImageLab software (Bio-Rad), the background was calculated for each spot from the

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median value of the surrounding baseline.

AFFINITY PURIFICATION

The soluble protein fraction was added to 100µl TALON of His-Tag affinity resin (Clontech). After 2h incubation at 4°C, the beads were washed 3 times with 2 Column

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Volumes (CV) of PBS 1x, 10mM Imidazole (Santa-Cruz), 2 CMC (critical Micelle concentration) of detergent. Elution was performed with 5-column volume of the

PAGE and western-blot. NATIVE-PAGE (CN-PAGE)

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same buffer with 200mM Imidazole. Samples of each fraction were analyzed by SDS-

Purified MP-A or MP-B (20µl) was separated on a Tris glycine TGX 4-15% acrylamide gel (BioRad) using ice-cold anode (25mM imidazole pH 8.0) and cathode

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(50mM Tricine, 0.05% Na-deoxycholate, 0.01 % DDM, 7.5 mM imidazole) buffers. Migration was performed for 70 min at 200V and analyzed by western-blot using specific and His-tag antibodies for MP-A and MP-B, respectively.

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ELECTRON MICROSCOPY

3 µl of each protein sample concentrated at 10µg/ ml was adsorbed on negative stain

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grid similarly to what was previously reported [38]. Typically, 200 Mesh copper grids coated with formvar-C were stained with 1% uranyl acetate and observed on a transmission electron microscope (Jeol 1400 JEM) equiped with a Gatan camera (Orius 600) and Digital Micrograph Software.

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LEGENDS Figure 1: Schematic representation of the novel systematic membrane proteins solubilization method (BMSS for Biotinylated Membranes Solubilization and Separation). 4 steps are described. 1-Insertion of biotinylated lipids into biological membranes. 2- Isolation of biotinylated biological membranes using streptavidine

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magnetic beads after sucrose gradient. 3- Membrane protein solubilization using detergents above the critical Micelle Concentration (10CMC). 4- Solubilized membrane proteins separation.

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Figure 2: Quality controls of the biotinylated membranes preparation.

A- Monitoring biotinylated lipids insertion into biological membranes and presence of

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membrane proteins of interest (MP-A or MP-B). Verification of the presence of MPA and MP-B after biotinylated membranes preparation in presence and absence of SDS, using magnetic streptavidin beads separation (B) or classical ultracentrifugation (C).

Figure 3: Use of BMSS method for small set of solubilization screening.

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A- Table of solubilization conditions performed for MP-A expressed in insect cells. Classical detergents (blue), calixarene based detergents (green) and combination conditions (gray) were indicated. B- Results obtained by dot blots (two streptavidin

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linked magnetic beads separation experiments and two dot blots for each experiment). Figure 4: Use of BMSS method for large set of solubilization screening.

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A- Table of the solubilization conditions performed for MP-A (from insect cells) and MP-B (from E.coli). Classical detergents (blue), calixarene based detergents (green) and combination conditions (gray) were indicated. B- Results obtained by dot blots on MP-A solubilization. C- Results obtained by dot blots on MP-B solubilization. Figure 5: Comparison between classical ultracentrifugation and BMSS method. SDS-PAGE and western blot analysis on total, soluble and insoluble fractions after ultracentrifugation of solubilized materiel using 17 representative conditions (good and bad corresponding to more or less than 50% solubilization rate, respectively) found by BMSS for MP-A (A) and 10 conditions for MP-B (B) using specific

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antibodies against each protein. * Potential non-dissociated oligomers. Good and bad conditions are represented in red and blue, respectively. The band quantification is shown in tables S5 and S6.

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Figure 6: Purification and characterization of MP-A and MP-B in solubilization conditions identified by BMSS.

A- Solubilization and affinity purification of MP-A and MP-B. The steps were monitored by SDS-PAGE followed by western blot and coomassie staining for MP-A

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and MP-B, respectively. T, P and S correspond to Total, pellet (insoluble) and Soluble fractions, respectively. B-Native PAGE of affinity purified MP-A and MP-B to

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evaluate the protein homogeneity and the aggregation status. C-Negative staining Electron microscopy images of purified MP-A and MP-B. *Potential non- dissociated

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oligomers.

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AKNOWLEDGMENTS: We wish to thank Emmanuel Dejean for his continuous support and discussion about the molecular basis of solubilization, Pierre Falson for providing us with MP-2 transporter plasmid, Anne Champagne for constant technical help. Kelly Garnier

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Thomas Iwema & Vincent Corvest and CALIXAR team for support.

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REREFENCES

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19. Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14: 673-681. 20. Bird LE, Rada H, Verma A, Gasper R, Birch J, et al. (2015) Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli. J Vis Exp: e52357. 21. Sonoda Y, Newstead S, Hu NJ, Alguel Y, Nji E, et al. (2011) Benchmarking membrane protein detergent stability for improving throughput of highresolution X-ray structures. Structure 19: 17-25. 22. Pullara F, Guerrero-Santoro J, Calero M, Zhang Q, Peng Y, et al. (2013) A general path for large-scale solubilization of cellular proteins: from membrane receptors to multiprotein complexes. Protein Expr Purif 87: 111-119. 23. Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, et al. (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3: 784-798. 24. Agharkar A, Rzadkowolski J, McBroom M, Gonzales EB (2014) Detergent screening of the human voltage-gated proton channel using fluorescencedetection size-exclusion chromatography. Protein Sci 23: 1136-1147. 25. Gutmann DA, Mizohata E, Newstead S, Ferrandon S, Postis V, et al. (2007) A high-throughput method for membrane protein solubility screening: the ultracentrifugation dispersity sedimentation assay. Protein Sci 16: 14221428. 26. Vergis JM, Purdy MD, Wiener MC (2010) A high-throughput differential filtration assay to screen and select detergents for membrane proteins. Anal Biochem 407: 1-11. 27. Schmidt A, Spinke J, Bayerl T, Sackmann E, Knoll W (1992) Streptavidin binding to biotinylated lipid layers on solid supports. A neutron reflection and surface plasmon optical study. Biophys J 63: 1385-1392. 28. Lundquist A, Hansen SB, Nordstrom H, Danielson UH, Edwards K (2010) Biotinylated lipid bilayer disks as model membranes for biosensor analyses. Anal Biochem 405: 153-159. 29. Davidson WS, Ghering AB, Beish L, Tubb MR, Hui DY, et al. (2006) The biotincapture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins. J Lipid Res 47: 440-449. 30. Rosati A, Basile A, D'Auria R, d'Avenia M, De Marco M, et al. (2015) BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages. Nat Commun 6: 8695. 31. Matar-Merheb R, Rhimi M, Leydier A, Huche F, Galian C, et al. (2011) Structuring detergents for extracting and stabilizing functional membrane proteins. PLoS One 6: e18036. 32. Cross TA, Sharma M, Yi M, Zhou HX (2011) Influence of solubilizing environments on membrane protein structures. Trends Biochem Sci 36: 117-125. 33. Janmey PA, Kinnunen PK (2006) Biophysical properties of lipids and dynamic membranes. Trends Cell Biol 16: 538-546.

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34. le Maire M, Champeil P, Moller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508: 86111. 35. Moller JV, le Maire M (1993) Detergent binding as a measure of hydrophobic surface area of integral membrane proteins. J Biol Chem 268: 1865918672. 36. Aloia AL, Glatz RV, McMurchie EJ, Leifert WR (2009) GPCR expression using baculovirus-infected Sf9 cells. Methods Mol Biol 552: 115-129. 37. Krettler C, Reinhart C, Bevans CG (2013) Expression of GPCRs in Pichia pastoris for structural studies. Methods Enzymol 520: 1-29. 38. Jawhari A, Uhring M, De Carlo S, Crucifix C, Tocchini-Valentini G, et al. (2006) Structure and oligomeric state of human transcription factor TFIIE. EMBO Rep 7: 500-505.

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Figure 1

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+

Incubation

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Sucrose gradient

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1-Insertion of biotinylated lipids into biological membranes

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2-Isolation of biotinylated biological membrane

detergent addition

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solubilized proteins

3-Membrane proteins solubilization

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+

4-Solubilized membrane proteins separation

+

solubilized proteins Non-solubilized proteins and membranes

Membrane proteins Streptavidine magnetic detergent beads

lipids biotinlylated lipids

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Membranes biotinylation

A

SC

Membranes

Antibody @MP-A or MP-B

Streptavidin linked magnetic beads incubation time (min)

Separation

Antibody@MP-A 60

AC C

(-)

30

120

Antibody@MP-B 15

30

60

120

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15 SDS Solubilization

MP-B (Transporter)

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MP-A (GPCR)

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Antibody @ Biotin

B

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Figure 2

(+)

MP-A (GPCR)

C Ultracentrifugation Antibody @ MP-A @ MP-B SDS Solubilization

(-) (+)

MP-B (Transporter)

C D

(-)

SDS

3 CALX153 ACE

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2

4

CALX163 ACE

5

DDM

CALX173 ACE

OG

6

B

CALX173 ACE/DM

CALX173 CALX1103 ACE/ OG ACE/OG

CALX173 GLUK

C12E8

CALX173 CALX1103 ACE/C12E8 ACE/C12E8

CALX183 ACE

DDM

CALX173 CALX1103 ACE/DDM ACE/DDM

1 E

CALX193 ACE

CHAPS

CALX173 CALX1103 ACE/CHAPS ACE/CHAPS

F

CALX1103 ACE

LDAO

CALX173 CALX1103 ACE/LDAO ACE/LDAO

CALX1113 ACE

FC12

G H

CALX1123 ACE

Sarkosyl

CALX173 CALX1103 ACE/FC12 ACE/FC12 CALX173 ACE/ Sarkosyl

CALX1103 ACE/ Sarkozyl

2

1

A B C D E F G H 2

2

3 4 5 6

2

3 4 5 6

1

3 4 5 6

SEPARATION2/ DOT1

B

1

AC C

A

SOLUBILIZATION CONDITIONS

3 4 5 6

1 A B C D E F G H

SEPARATION2/ DOT2

A

SEPARATION1/ DOT1

Figure 3

SEPARATION1/ DOT2

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A

SC

Figure 4

SOLUBILIZATION CONDITIONS

A

2

(-)

B

CALX163 ACE

CALX183 ACE

CALX153 ACE

CALX173 ACE

CALX133 ACE

D

CALX153 CALX173 CYMAL-5 ACE ACE / CYMAL-5 /CYMAL-5

F

OG

G

LMNG

H

DM

2

3

4

CALX193 ACE

CALX111 ACE

6(-)

SDS 7

8

9

10

11

12

CALX153 CALX173 CALX193 CALX1113 CALX173 ACE/DDM ACE/DDM ACE/DDM ACE/DDM GLUK/DDM

DDM

CALX153 CALX173 Digitonin ACE/ ACE/ Digitonin Digitonin

CALX193 ACE/ Digitonin

CALX1113 CALX173 ACE/ GLUK/ Digitonin Digitonin

CALX1113 CALX173 CALX153 CALX173 CALX193 CALX173 Triton X100 ACE/ Triton ACE/ Triton ACE/ Triton ACE/ Triton GLUK/ GLUK X100 Triton X100 X100 X100 X100

CALX193 CALX111 CALX173 GLUK ACE ACE /CYMAL-5 /CYMAL-5 /CYMAL-5

CHAPS

CALX153 ACE/ CHAPS

CALX173 ACE/ CHAPS

CALX193 ACE/ CHAPS

CALX1113 CALX173 ACE/ GLUK/ CHAPS CHAPS CALX1113 CALX173 ACE/ GLUK/ CHAPS CHAPS

LDAO

CALX153 ACE/ CHAPS

CALX173 GLUK/OG

FC12

CALX153 CALX173 CALX193 CALX1113 CALX173 ACE/FC12 ACE/FC12 ACE/FC12 ACE/FC12 GLUK/FC12

CALX173 CALX153 CALX173 CALX193 CALX111 GLUK/ ACE/LMNG ACE/LMNG ACE/LMNG ACE/LMNG LMNG

FC14

CALX153 CALX173 CALX193 CALX1113 CALX173 ACE/FC14 ACE/FC14 ACE/FC14 ACE/FC14 GLUK/FC14

CALX153 ACE/DM

Sarkosyl

CALX153 ACE/ Sarkosyl

CALX173 CALX153 CALX173 CALX193 CALX111 GLUK/ ACE/C12E8 ACE/C12E8 ACE/C12E8 ACE/C12E8 C12E8 CALX153 ACE/OG

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C12E8

5

CALX1103 CALX1123 CALX1163 ACE ACE ACE

CALX173 ACE/OG

CALX193 ACE/OG

AC C

E

4

CALX173 DDM/ CALX DDM/ CALX DDM/ CALX 1103ACE 1103ACE ACE (2CMC)173ACE (10CMC) (5CMC) (2CMC)

C

B 1

SDS

3

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1

TE D

(-)

CALX173 ACE/DM

CALX193 ACE/DM

CALX111 ACE/OG

CALX111 CALX173 ACE/DM GLUK/DM

MP-A (GPCR) 5 6 7 8 9 10 11 12

1 A

B

B

C

C

D

D

E

E

F

F

G

G

H

H

CALX173 ACE/ Sarkosyl

CALX193 ACE/ CHAPS

CALX193 ACE/ Sarkosyl

CALX1113 CALX173 ACE/ GLUK/ Sarkosyl Sarkosyl

MP-B (Transporter)

C

A

CALX173 ACE/ CHAPS

2

3

4

5

6

7

8

9 10 11 12

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A kDa

(-)

CALX173

T P S T

P S

CALX183

CALX193

T P S

T P

CALX1113

OG

LMNG

T P S

T P S T P S T P S

S

LMNG/ CALX173

SC

T P S T P S

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100 70 55 40 25 15

2 3

4

LDAO

T P S

5

6

7 8 9 10 11 12

LDAO/ CALX173

LDAO/ CALX1113

T

T P S

P S

FC14/ CALX174

FC14

T P

250 170 140

Sarkosyl

S T P

S T P

40

MP-A

25 26 27 28 29 30

S T P

S

* * MP-A

AC C

25

* *

Sarkosy/ CALX173

EP

100 70 55

13 14 15 16 17 18 19 20 21 22 23 24

TE D

1

15

31 32 33 34 35 36 37 38 39

B (-)

kDa

DDM/ CALX173

DDM

250 170 140

kDa

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Figure 5

CALX153

T P S

T

CALX173

T P S

P S

40 41 42 43 44 45 46 47 48 49 50 51

CALX1113

T P

S

DDM

DDM/ CALX153

Triton

Triton/ CALX173

CHAPS

T P S

T P S

T P S

T P S

T P S

FC14

T

P S

250 170 140

*

100 70

MP-B

55 40 25 15 1

2 3

4

5

6

7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 23 24 25 26 27 28 29 30

kDa

T P

S

250 170 140

T P

S

MP-A

Nu-PAGE

E1 E2 E3 E4 E5

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*

40

100 70 55

1

2

* MP-B

40 25

EP

25

15

15 4 5

6

7 8

1 2 3

4

5

6

7 8

AC C

1 2 3

C

kDa

E1 E2 E3 E4 E5

250 170 140 100 70 55

B

SDS-PAGE/ Coomassie

SDS-PAGE/ WB

MP-B

A

MP-A

Figure 6

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Electron Microscopy

MP-A

MP-B