The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution
Membrane proteins play essential roles , such as membrane transport , signal transduction , cell homeostasis , and energy metabolism . Despite their importance , obtaining these proteins in a stable non-aggregated state remains problematic . Membrane proteins are generally purified in detergent micelles , but these small amphipathic molecules are quite often detrimental to protein structure and activity , in addition to interfering with downstream analytical methods . This drawback has led researchers to develop detergent-free alternatives such as amphipols ( Popot , 2010 ) , SMALPs ( Lee et al . , 2016 ) , saposin-lipoparticles ( Frauenfeld et al . , 2016 ) , and the popular nanodisc system ( Bayburt et al . , 2006; Denisov et al . , 2004 ) . In the latter case , two amphipathic membrane scaffold proteins ( MSPs ) derived from apoA1 wrap around a small patch of lipid bilayer containing the target membrane protein ( Bayburt et al . , 2006; Denisov et al . , 2004; Denisov and Sligar , 2016 ) . However , in spite of an apparent simplicity , the formation of a nanodisc depends on several factors such as lipid to protein ratio , scaffold length , nature of lipids , rate of detergent removal and overall amenability of the target for re-assembly into lipid bilayer ( Bayburt et al . , 2006; Denisov et al . , 2004; Hagn et al . , 2013 ) . The method is therefore not trivial and small deviations from optimal conditions often leads to low-efficiency reconstitution , or else liposome formation or protein aggregation ( Bayburt et al . , 2006 ) . Peptides have been considered as an alternative to scaffold proteins . Peptergents ( Corin et al . , 2011 ) , lipopeptides ( Tao et al . , 2013 ) , nanostructured [beta]-sheet peptides ( Privé , 2009 ) , and bi-helical derivatives of the ApoA1-mimetic 18A peptide , termed ‘beltides’ ( Larsen et al . , 2016 ) , have been reported in the recent years . Yet , these systems have not been widely adopted for structural or functional characterization of membrane proteins for various reasons . Peptergents and lipopeptides can solubilize membrane proteins directly from a lipid bilayer , but same as detergent these peptides form mixed micelles that readily aggregate and precipitate below a certain critical micellar concentration ( Corin et al . , 2011; Tao et al . , 2013 ) . Beltides were shown to trap bacteriorhodopsin in solution , but the method required prior incubation with specific amounts of lipids and the particles formed were reportedly unstable at physiological temperatures ( Larsen et al . , 2016 ) . Cost and complexity of the peptide can also be problematic . Nanostructured [beta]-sheet peptides contain extended alkyl chains covalently linked to glycine residues , while lipopeptides need to be covalently linked to a lipid molecule ( Tao et al . , 2013; Privé , 2009 ) . These hydrophobic peptides are also difficult to work with given their low solubility . Peptergents require titration of base to become soluble in aqueous solution ( Corin et al . , 2011 ) , and [beta]-sheet peptides can form extended filament clusters without detergent ( Privé , 2009 ) . Thus , a peptide-based reconstitution method that is cost-effective , rapid , unhindered by issues of solubility and generally applicable to membrane proteins remains to be developped . We present the peptidisc . The peptidisc is made by multiple copies of an amphipathic bi-helical peptide ( hereafter termed NSPr ) wrapping around its target membrane protein . In contrast to the nanodisc , no additional lipids are necessary during reconstitution , except those that have co-purified with the protein ( i . e . annular lipids ) . The peptide design we employ is a reverse version of the original nanodisc scaffold peptide ( NSP ) , which consists of two repeats of the ApoA1-derived 18A peptide joined by a flexible linker proline ( Kariyazono et al . , 2016; Chung et al . , 1985 ) , in addition to two leucine residues which are substituted by phenylalanines to increase lipid affinity ( Kariyazono et al . , 2016; Mishra et al . , 2008 ) ( Supplementary file 1 ) . The original NSP can stabilize lipid particles ( Kariyazono et al . , 2016 ) , but issues of water-solubility and adaptability to membrane proteins were not demonstrated . We show here the NSPr design is very effective for stabilizing both α-helical and β-barrel membrane proteins of different size , topology , and complexity .
The original NSP is able to capture lipids from a lipid bilayer and forms discoidal particles of varying size ( Kariyazono et al . , 2016 ) . However , due to its hydrophobicity , the NSP peptide is difficult to solubilize; the solution is cloudy after resuspension in water ( Figure 1B ) . To increase peptide solubility , we reversed the amino acid sequence of NSP , so that the two amphipathic helices have a slightly altered orientation , leading to a lower hydrophobic moment and higher overall electropotential ( Figure 1AC , Supplementary file 1 ) . We also forewent acetylation and amidation of the peptide termini so that modifications such biotinylation remain possible . These small modifications allowed the final design ( termed NSPr ) to be fully dissolved in water at concentrations up to 25 mg/ml ( Figure 1B ) . We tested the ability of NSPr to capture the ABC transporter MalFGK2 using an ‘on-column’ reconstitution method ( Figure 2 ) . The NSPr peptide was mixed with MalFGK2 in dodecyl maltoside and the mixture applied immediately onto a size exclusion column equilibrated in a detergent-free buffer ( Figure 2A ) . The collected particles ( hereafter termed peptidisc or MalFGK2-NSPr ) were soluble and monodisperse , as shown by clear-native CN-PAGE and blue-native BN-PAGE ( Figure 2B ) . We determined the approximate peptide content using SDS-PAGE ( Figure 3A ) . Also , since annular lipids are tightly bound to membrane proteins ( Bechara et al . , 2015 ) , we determined the final lipid content in the peptidisc using thin layer chromatography and photocolorimetric methods ( Figure 4A and B ) . This analysis indicated a stoichiometry of 10 ± 2 peptides and 41 ± 10 lipids per MalFGK2 ( Table 1 ) . This stoichiometry allowed us to calculate the mass of the MalFGK2 peptidisc to 251 ± 12 kDa ( Table 1 ) . Interestingly , the lipids identified in the TLC analysis were predominantly negative phospholipids , cardiolipin and phosphatidylglycerol ( Figure 4B ) . These lipids play a role in regulation of MalFGK2 by stabilizing interactions with the regulatory protein EIIA ( Bao and Duong , 2013 ) . To corroborate the peptide and lipid stoichiometry , we determined the molecular weight of the intact complex by native mass spectrometry ( 247 ± 24 kDa; Table 1 , Figure 5A and B ) , and size exclusion chromatography coupled multi-angle light scattering ( SEC-MALS ) ( 250 ± 17 kDa; Table 2 , Figure 6A ) . The SEC-MALS analysis showed that the peptidisc remains perfectly stable during storage ( e . g . 3 days at 4°C ) . We then examined the particles by single particle negative-stain electron microscopy ( Figure 2C ) . The 2D-class averages revealed a structure very similar to MalFGK2 in nanodiscs , ( Fabre et al . , 2017 ) with distinctly visible elements such as the MalK2 dimer , the periplasmic P2 loop and a larger discoidal density corresponding to the NSPr peptides wrapping around the MalFG membrane domain . The measured diameter of the peptidisc was 11 . 7 ± 1 . 4 nm , which is consistent with a stoichiometry of 12 ± 2 peptides per MalFGK2 complex when arranged in the double-belt model ( Table 2 ) . Finally , the ATPase activity of MalFGK2 in peptidisc was similar to that reported in proteoliposomes and in nanodiscs ( Bao and Duong , 2012 ) , in sharp contrast to the high and unregulated ATPase activity observed in detergent micelles ( Figure 2D ) . Importantly , the structural integrity of the peptidisc remained stable at elevated temperatures , with ~80% of MalFGK2 peptidisc intact after incubation for 3 hr at 30°C ( Figure 6B ) . We also incorporated β-barrel membrane proteins in peptidisc , using the FhuA receptor as a model protein ( Figure 7A ) . Analysis of FhuA-peptidisc by native mass spectrometry indicated a molecular weight of 138 ± 17 kDa ( Figure 5E and F ) . Quantitation of the individual peptide and lipid components , following the approach applied to MalFGK2 above , indicated an average of 8 ± 3 lipids and 10 ± 2 NSPr per FhuA ( Table 1 , Figure 3B and Figure 4A ) . These measurements were used to estimate a molecular mass of 131 ± 9 kDa ( Table 1 ) . Binding analysis on CN-PAGE further showed that FhuA in peptidisc is functional for both TonB and colicin M ( Figure 7B ) . The binding of TonB and colicin M is modulated by the ligand ferricrocin ( Figure 7B ) , as previously reported in vivo and in vitro ( Mills et al . , 2014; Wayne et al . , 1976 ) . The peptidisc is therefore suitable for the functional reconstitution of both α-helical and β-barrel membrane proteins . The ‘in-gel’ method ( Figure 8 ) was developed to determine optimal reconstitution conditions in a time- and cost-effective manner . Small amounts of peptides ( 0–2 . 5 µg ) were mixed with the target protein ( ~1 . 25 µg ) in detergent solution , and the resulting mixture immediately loaded on native gel . In that case , removal of the non-ionic detergent occurs during electrophoresis when the protein-peptide mixture enters the detergent-free part of the gel . At the correct NSP ratio , the target membrane protein does not aggregate at the top of the gel but instead migrates in a soluble form to its expected molecular weight position . This simple method allowed us to estimate the effective NSP concentrations required to trap four different integral membrane complexes into a peptidisc: MalFGK2 ( Figure 8A ) , FhuA ( Figure 8B ) , the trimeric OmpF porin ( Figure 8C ) , and the membrane translocon SecYEG ( Figure 8D ) . These membrane proteins were generally reconstituted at similar peptide concentrations , with a half-maximal molar ratio ( RR50 ) of 20 ( Figure 8E and F ) . This is significantly higher than the measured stoichiometry of ~10 peptides per protein complex ( Table 1 ) , suggesting that excess peptide is needed to achieve efficient assembly . This analysis also showed that the SecYEG complex can be trapped as a dimer and higher order oligomeric form in peptidisc ( Figure 8D ) , probably due to the self-association of this complex in detergent solution , as shown before ( Bessonneau et al . , 2002 ) . This later observation further differentiates the peptidisc from the nanodisc . In the nanodisc , the selective reconstitution of the SecYEG monomer and dimer requires MSP proteins of different lengths ( Figure 8—figure supplement 1 ) ( Dalal et al . , 2012 ) . To save on peptide consumption , as well as to minimize exposure of the target to detergent , we developed the ‘on-beads’ reconstitution method ( Figure 9 ) , wherein affinity-purification of the protein and incorporation into peptidisc are carried out simultaneously . As illustrated in Figure 9A , the peptide was added in excess while the membrane protein still bound to the beads , followed by detergent dilution and eventually elution in a detergent-free buffer ( Figure 9A , step 5 ) . The method was tested using the his-tagged MalFGK2 complex ( Figure 9B ) . Analysis of the eluted complex by BN-PAGE and CN-PAGE showed that MalFGK2 is readily incorporated into peptidiscs ( Figure 9C ) , with purity and yield as good as with conventional detergent-based chromatography ( Figure 9C ) . As an added advantage , the excess peptide collected before the elution step was re-used for the next round of ‘on-beads’ reconstitution , thereby reducing the cost of large-scale reconstitution . Finally , we reconstituted the photosynthetic bacterial reaction center ( BRC ) from Rhodobacter sphaeroides , given its potential for biotechnological application . The BRC is employed in bio-hybrid solar cells due to its ability to absorb light in the near-infrared with high quantum efficiency ( Blankenship et al . , 2011; Ravi and Tan , 2015; Yaghoubi et al . , 2017 ) . However , sustained heat and light exposure lead to irreversible loss of pigments and protein denaturation ( Hughes et al . , 2006; Scheidelaar S et al . , 2014 ) . Thermal stability and solubility at high protein concentration are therefore important parameters for successful application . The molecular weight of the BRC peptidisc measured by native mass spectrometry is 138 ± 18 kDa ( Table 1 , Figure 5C and D ) . It has been shown that purification of BRC in LDAO delipidates the complex ( Scheidelaar S et al . , 2014 ) , and accordingly the BRC peptidisc contained only 4 ± 1 phospholipids ( Table 1 , Figure 4A ) . Analysis by SDS-PAGE indicated a stoichiometry of 9 ± 1 NSP per BRC ( Table 1 , Figure 3C ) . The calculated molecular weight was 138 ± 5 kDa , in excellent agreement with native mass spectrometry data ( Table 1 ) . We next measured the stability of the BRC pigments in peptidiscs or in LDAO detergent ( Figure 10—figure supplement 1A and B ) . The spectral properties of the BRC complex were similar in both environments ( Figure 10A ) . However , the BRC in peptidisc resisted denaturation 65°C for 1 hr , while it was fully denatured in less than 4 min in LDAO ( Figure 10B ) . This difference corresponds to ~100 fold increase of the half-life of the BRC complex at elevated temperatures ( Figure 10C ) . We also compared the thermal stability of the BRC complex when reconstituted without additional lipids in SMA polymer and nanodiscs ( Figure 10—figure supplement 1C and D ) . Without added lipid , the diameter of the BRC is too small for the MSP1D1 belt , resulting in some protein aggregation and heterogenous nanodisc preparation ( Figure 10—figure supplement 1F , left panel , lane 4 ) . Reconstitution into the SMA polymer without lipids was monodisperse ( Figure 10—figure supplement 1F , left panel , lane 5 ) and the thermostability was higher than in LDAO ( Figure 10—figure supplement 1E ) . This observation is contrary to previous reports which suggest that the lipid environment in the SMA particle is what increases BRC thermostability . ( Scheidelaar S et al . , 2014 ) In our hands , reconstitution of the BRC into peptidiscs , proteoliposomes , low-lipid nanodiscs and SMA particles all result in comparable thermostability ( Figure 10—figure supplement 1E ) . From these results , it appears more important to remove detergents than include lipids to increase thermal tolerance of the BRC .
Detergents remain the most effective way to extract and purify membrane proteins , yet these surfactants have many undesired effects on protein stability and downstream biochemical analysis . To circumvent these difficulties , and to handle these proteins like their water-soluble counterparts , membrane proteins are more often reconstituted with amphipathic scaffolds . However , current methods of reconstitution are difficult because each scaffold system has specific properties and limitations , and each requires substantial optimization . The aim of our work was to develop a ‘one-size fits all’ method to streamline the capture of membrane proteins in detergent-free solution . We show that the peptidisc is a simple and efficient way to replace detergent . The system works with membrane proteins of different size , fold and complexity , and does not require addition of exogenous lipids . The peptidisc captures membrane proteins regardless of their initial lipid content , and therefore the use of exogenous lipids to match the diameter of the scaffold such as in the nanodisc system is avoided . Since the binding of the peptide is essentially guided by the size and shape of the protein template , the peptide stoichiometry is also self-determined . As a direct consequence , the preparation of peptidisc is possible through rapid detergent removal techniques such as ‘in-gel’ , ‘on-column’ and ‘on-bead’ methods . Each of these methods has considerable advantages compared to overnight dialysis and ‘biobeads’ techniques traditionally used for scaffold reconstitution . The ‘in-gel’ method is fast , high throughput and requires only 1–2 µg of the precious target protein in order to identify the optimal peptide ratio ( Figure 8 ) . The ‘on-column’ method allows direct preparation of large quantities of peptidiscs with simultaneous removal of salts , detergent and excess peptides . However , gel filtration requires concentrated protein samples and unbound peptide is wasted . The ‘on-bead’ method is therefore ideal for the peptidisc reconstitution because protein purification , detergent removal , trapping in peptidisc and recovering of unbound peptide are carried out simultaneously in the same tube . Additionally , because long exposure or storage of the protein in detergent is avoided , the ‘on-bead’ method can be especially advantageous in the case of unstable membrane proteins . Beside these methodological considerations , we show that the peptidisc maintain proteins in a functional state . For instance , both FhuA and MalFGK2 retain their ability to interact with their soluble binding partners . In contrast to the detergent , the ATPase activity of MalFGK2 in peptidisc regains dependence to substrate and maltose-binding protein MalE , indicating a return to the transporter’s native membrane conformation ( Bao and Duong , 2012 ) . Negative-stain electron microscopy of MalFGK2 shows good resolution for the periplasmic P2 loop and cytosolic ABC domains , whereas the transmembrane domains MalFG are surrounded by an extra density corresponding to the peptide belt and naturally present lipids ( Figure 2C ) . Lipid quantitation indeed indicates that ~ 40 lipids molecules , mostly acidic , are captured with MalFGK2 ( Table 1 , Figure 4A ) . In the case of the BRC complex however , there must be direct protein-peptide contact because almost no lipids are detected in the final assembly ( Table 1 , Figure 4A and B ) . Despite the absence of lipids , the thermal stability of the BRC in peptidisc is still much higher than in detergent , with a melting temperature similar to that observed proteoliposomes ( Figure 10—figure supplement 1E ) . Clearly , the peptidisc is more than a simple surrogate of the detergent molecules; the peptide assembly forms an environment that stabilize and support the transmembrane domains of a membrane protein from aqueous solution . The peptidisc offers distinct advantages when compared to other amphipathic scaffolds . Unlike beltides , peptergents , lipopeptides , and nanostructured [beta]-sheet peptides , the NSPr does not require modifications at its N- or C-termini ( Corin et al . , 2011; Tao et al . , 2013; Privé , 2009; Larsen et al . , 2016 ) . Both ends of NSPr remain available for modifications , such as biotinylation , which we show does not affect peptidisc assembly ( Figure 3B ) . Importantly , peptergents and lipopeptides have critical micelle concentrations and dynamically exchange in solution . Because of this instability , an excess of these peptides is always needed to keep membrane proteins in solution ( Corin et al . , 2011; McGregor et al . , 2003 ) . This is not the case for the peptidisc because NSPr binds strongly to its target , allowing free peptides to be removed without compromising peptidisc stability . The length of the NSPr also does not need to be adjusted to the diameter of the target protein . This property may be especially advantageous when capturing macromolecular membrane protein complexes or proteins that exist in oligomeric state , as is the case with the SecYEG complex ( Figure 8—figure supplement 1B ) . Lastly , the NSPr is synthetically made and can be obtained in large quantity , high purity and free of immunogens usually found in recombinant cell expression systems ( Schwarz et al . , 2014 ) . The structural homogeneity of NSPr is also high compared to other synthetic scaffolds , such as amphipols and styrene-maleic acids ( SMA ) . This is because peptide synthesis is sequential , whereas addition of carboxylate and other repeating units in amphipols and industrially prepared SMA polymers is randomly distributed along the polymer chain during assembly . Due to the ‘one-pot’ synthesis , these synthetic polymer preparations are often polydisperse mixtures of different lengths ( Popot , 2010; Smith et al . , 2017 ) . This compositional heterogeneity can be problematic for functional and structural studies ( Deller et al . , 2016 ) . Finally , due to both positive and negative charged amino acids , the solubility of the NSPr remains high at various pH or with divalent cations . Both these conditions can destabilize assemblies formed by synthetic polymers because they largely depend on charged carboxylate groups for solubility ( Popot , 2010; Gulati et al . , 2014 ) . How exactly the peptidisc wraps around the membrane protein template remains an important question . In ApoA1 lipid nanodiscs , the two scaffold proteins arrange themselves in an anti-parallel ‘double belt’ configuration ( Bibow et al . , 2017 ) . If NSPr were also arranged in a double belt , then the peptide to protein ratio would expectedly vary by factor of 2 , as an odd number of scaffold would leave part of the protein exposed to the environment . However , the native mass spectrometry profiles for FhuA and BRC indicate peptidisc populations which can differ in mass by one peptide only ( Figure 5D and F ) , suggesting an arrangement that is flexible . Possibly , the NSPr could be arranged in an orthogonal ‘picket fence’ orientation as proposed for lipopeptides , nanostructured [beta]-sheet peptides , and single helix ApoA1 mimetic peptides . ( Tao et al . , 2013; Privé , 2009; Islam et al . , 2018 ) . However , the length of NSPr ( 37 amino acids ) is too long to be orthogonal while maintaining contact with hydrophobic parts of the protein or alkyl chains of annular lipids . Thus , the NSPr perhaps simply lies in a tilted orientation . A tilted orientation would facilitate optimal binding and stoichiometry as the peptide shifts in angle of association , adapting to best fit the target membrane protein template . In conclusion , the peptidisc offers several advantages . The method is cheap , fast and seamlessly integrated in existing protein purification protocols , such as size exclusion and affinity chromatography . The peptide is relatively simple to synthesize and it can be recycled via the ‘on-bead’ method to decrease consumption . The peptidisc is not hindered by issue of buffer instability or heterogeneity as observed with other synthetic scaffold . Since the peptide self-associates to its template and without added lipids , this could be advantageous for structural studies which are affected by compositional heterogeneity . There are of course applications where other scaffold systems are better suited , such as direct protein solubilization with SMA polymers , or control over lipid environment offered by nanodiscs . Nevertheless , the current advantages of the peptidisc surely diminish the challenges associated with biochemical , structural and pharmacological characterization of purified membrane proteins . The peptidisc may emerge as a very practical way to analyse or exploit membrane proteins in a detergent-free environment .
Tryptone , yeast extract , NaCl , imidazole , Tris-base , acrylamide 40% , bis-acrylamide 2% and TEMED were obtained from Bioshop , Canada . Isopropyl β-D-1-thiogalactopyranoside ( IPTG ) , ampicillin , and arabinose were purchased from GoldBio . Detergents n-dodecyl-β-d-maltoside ( DDM ) and octyl-β-D-glucoside ( β-OG ) were from Anatrace . Detergent N , N-dimethyldodecylamine N-oxide ( LDAO ) was from Sigma . Total E . coli lipids were purchased from Avanti Polar Lipids . Resource 15Q , Fast Flow S , Superdex 200 HR 10/300 GL and 5/150 GL were obtained from GE Healthcare . Ni2+-NTA chelating Sepharose was obtained from Qiagen . All other chemicals were obtained from Fischer Scientific Canada . Peptide NSPr ( Nter-FAEKFKEAVKDYFAKFWDPAAEKLKEAVKDYFAKLWD-Cter ) and NSP ( Nter- DWLKAFYDKVAEKLKEAAPDWFKAFYDKVAEKFKEAF-Cter ) were obtained from A+ peptide Co . Ltd . and Genscript ( each with purity >80% ) . Peptide NSPrbio was obtained from KareBay . The effective purity of the peptides employed is actually higher as mass spectrometry analysis finds that main contaminants ( >10% ) consists of peptides missing the final aspartate residue . To aid accessibility to the academic community , bulk NSPr peptides and core protocols are available at www . peptidisc . com For solubility test experiments , lyophilized NSP and NSPr ( purity of 82% and 85% , respectively ) were resuspended in dH20 at room temperature to final concentrations of 15 mg/mL and 25 mg/mL , respectively . Peptide concentration was determined by absorbance at 280 nm . Residual TFA from peptide synthesis results in a low pH solution ( pH 2–3 ) . For all other experiments , peptides were solubilized in dH2O at 6 mg/mL . Solubilized peptides were stored at 4°C for up to 5 weeks . Immediately before use , the pH of the peptide solution was modified by addition of 20 mM Tris-HCl , pH 8 to form the so-called Assembly Buffer . Immediately before use in peptidisc reconstitutions , peptide concentration in Assembly Buffer was verified by Bradford assay ( Prehna et al . , 2012 ) . Unless otherwise stated , all proteins were expressed in E . coli BL21 ( DE3 ) ( New England Biolabs ) for 3 hr at 37°C after induction at an OD of 0 . 4–0 . 7 in LB medium supplemented with required antibiotic . Cells were harvested by low-speed centrifugation ( 10 , 000 x g , 6 min ) and resuspended in Buffer A ( 50 mM Tris-HCl: pH 8; 100 mM NaCl; 10% glycerol ) . Resuspended cells were treated with 1 mM phenylmethylsulfonyl fluoride ( PMSF ) and lysed using a microfluidizer ( Microfluidics ) at 10 , 000 psi . Unbroken cell debris and other aggregates were removed by an additional low-speed centrifugation . Cytosolic and crude membrane fractions containing the overexpressed protein of interest were subsequently isolated by ultracentrifugation ( 100 , 000 x g , 45 min ) and crude membrane fraction resuspended in Buffer A ( 50 mM Tris-HCl: pH8 , 100 mM NaCl , 10% glycerol ) . MalE and His-tagged MalFGK2 were purified as previously described ( Bao and Duong , 2012 ) , expressed from plasmids pBAD33-MalE and pBAD22-FGKhis , respectively . Crude membrane containing His-tagged MalFGK2 were solubilized at 4°C overnight in Buffer A + 1% DDM and clarified by ultracentrifugation . Solubilized MalFGK2 was isolated by Ni2+-chelating chromatography in Buffer A + 0 . 02% DDM , washed in five column volumes ( CV ) of Buffer B ( 50 mM Tris-HCl: pH 8; 200 mM NaCl; 15 mM imidazole; 10% glycerol ) +0 . 02% DDM , and then eluted in Buffer C ( 50 mM Tris-HCl: pH 8; 100 mM NaCl; 400 mM imidazole; 10% glycerol ) +0 . 02% DDM . Protein MalE was isolated on Resource 15Q column , concentrated using a 30 kDa polysulfone filter ( Pall Corporation ) , and then further purified on Superdex 200 HR 10/300 GL column equilibrated in Buffer EQ ( 50mM Tris-HCl: pH 8 , 50 mM NaCl , 10% glycerol ) . His-tagged-MSPL156 and His-tagged TonB23-329 were purified by Ni2+-chelating chromatography as previously described ( Mills et al . , 2014 ) . His-tagged Colicin M was expressed and purified according to established protocols from plasmid pMLD189 in the E . coli strain BW25113 ( Mills et al . , 2014 ) . His-tagged FhuA , encoded by plasmid pHX405 , was expressed in E . coli strain AW740 ( ΔompF , ΔompC ) in M9 minimal media and was purified in LDAO as previously described ( Mills et al . , 2014 ) OmpF was expressed from E . coli JW2203 ( ΔOmpC ) as previously described ( Jun et al . , 2014 ) . Prepared crude membrane was resuspended in Buffer A and the inner membrane solubilized by addition of 1% Triton X-100 . The outer membrane fraction ( OM ) was isolated by ultracentrifugation , resuspended in Buffer A + 1% LDAO at a concentration of 3 mg/mL , and incubated overnight at 4°C with gentle rocking . Insoluble material was removed by an additional ultracentrifugation step , and the clarified lysate was applied onto a Resource 15Q column pre-equilibriated in Buffer EQ + 0 . 1% LDAO . OmpF was eluted by a linear 20 mL gradient of 50–700 mM NaCl , and further purified by Superdex 200 HR 10/300 in Buffer A + 0 . 1% LDAO . Expression and purification of His-tagged SecYEG was performed from the plasmid pBad22-His-EYG as previously described ( Dalal et al . , 2012 ) . Crude membranes were solubilized for 1 hr at 4°C in Buffer A + 1% DDM . Solubilized material was clarified by ultra-centrifugation and passed over a 5 mL Ni2+-NTA column . After extensive washing in Buffer A + 0 . 02% DDM , SecYEG was eluted in Buffer A + 0 . 02% DDM over a 20 mL gradient of 0–600 mM imidazole . The most concentrated fractions were pooled and diluted fivefold in Buffer O ( 50 mM Tris-HCl: pH 8 , 10% glycerol +0 . 02% DDM ) before being applied to a 5 mL Fast Flow S cation exchange column pre-equilibrated in Buffer EQ + 0 . 02% DDM . Bound protein was eluted over a 20 mL gradient from 50 to 600 mM NaCl in Buffer EQ + 0 . 02% DDM . Plasmids pET28 encoding his-tagged MSPD1 and MSP1D1E3 proteins were transformed into BL21 cells and protein expression and purification was performed as previously described ( Dalal et al . , 2012 ) . All proteins , with the exception of BRC , were flash frozen in liquid nitrogen immediately after purification and stored at −80°C for later use . BRC was purified as previously described ( Bradford , 1976 ) . In brief , His-tagged BRC was expressed in Rhodobacter Sphaeroides RcX ( ΔpuhA , ΔpufQBALMX , ΔrshI , ΔppsR ) using plasmid pIND4-RC1 . A preculture of 10 mL in RLB media ( LB medium; 810 µM MgCl2; 510 µM CaCl2 ) +25 µg/mL kanamycin was transferred into 100 ml of RLB-kan and grown overnight at 30°C before transfer into 1 L of freshly prepared RLB-kan . After growth for 8 hr at 30°C , BRC production was induced with 1 mM IPTG for an additional 16 hr . During growth and purification , light exposure was kept to a minimum . Cells were harvested by low-speed centrifugation , resuspended in Buffer A and lysed by French press ( 10 , 000 psi ) . Unbroken cells and cell debris were removed by low speed centrifugation , and the supernatant treated with 1% LDAO overnight at 4°C . After removal of insoluble material by ultracentrifugation , the supernatant was supplemented with 10 mM imidazole and the BRC purified by Ni2+-chelating affinity chromatography . BRC bound to affinity resin was washed overnight at 4°C in 20 column volumes of Buffer B + 0 . 03% LDAO , before elution in Buffer C + 0 . 03% LDAO . The complex was further purified on a Superdex 200 HR 10/300 GL in Buffer A + 0 . 03% LDAO , and stored in the dark at 4°C before use in thermostability assays . MalFGK2 ( 300 µg ) in Buffer E + 0 . 02% DDM was mixed with NSP ( 480 µg ) in Assembly Buffer in a total volume of 100 µL . The mixture was immediately injected onto a 100 µL loop connected to a Superdex 200 HR 5/200 GL column running at 0 . 4 ml/min in Buffer AC ( 50 mM Tris-HCl , pH 8; 100 mM NaCl ) . Fractions were collected , pooled , concentrated using a 100 kDa polysulfone filter ( Pall Corporation , USA ) , and stored at 4°C . For on-column reconstitution of FhuA , 500 µL of the protein ( 1 mg ) was mixed with NSP ( 1 . 8 mg ) in Buffer A + 0 . 05% LDAO , and injected onto a 500 µL loop connected to a Superdex 200 HR 10/300 GL column running at 0 . 5 mL/min in Buffer AC . The target membrane protein ( ~1 . 25 µg ) was mixed with increasing concentrations of NSPr ( 0–2 . 5 µg ) and allowed to incubate for 1–2 min at room temperature . The mixture was then supplemented with Buffer A to bring the final detergent concentration below its CMC ( 0 . 008% and 0 . 01% for DDM and LDAO , respectively ) while keeping the final volume to 15 µL . A solution of glycerol was added to 10% final to facilitate loading on 4–12% CN-PAGE . The electrophoresis was set constant at 25mA for 1 hr at room temperature . Bands were visualized by Coomassie Blue G250 staining . Crude membranes ( 10 mL at 7 . 5 mg/ml total protein content ) containing overexpressed MalFGK2 were solubilized in Buffer A + 1% DDM for 1 hr at 4°C before removal of insoluble aggregate by ultracentrifugation ( 100 , 000 x g , 1 hr , 4°C ) . The solubilized membrane proteins were incubated with 200 µl of Ni-NTA resin ( Qiagen ) pre-equilibriated in Buffer A + 0 . 02% DDM for 1 hr at 4°C . The Ni-NTA beads were collected by low-speed centrifugation ( 3000 x g , 3 min ) , washed twice with 10 CV of Buffer B supplemented with 0 . 02% DDM . Post-washing , 10 CV of Assembly Buffer ( 1 mg/mL NSPr in 20 mM Tris-HCl pH 8 ) was added to the beads and allowed to incubate for 5 min on ice . The Assembly Buffer was removed and the beads loaded into a gravity column with 10 CV of Buffer B ( 50 mM Tris-HCl , pH 8; 200 mM NaCl , 10% glycerol , 15 mM imidazole ) . The assembled peptidiscs were subsequently treated with 500 µL of Buffer C ( 50 mM Tris-HCl , pH 8; 100 mM NaCl;10% glycerol; 400 mM imidazole ) to elute the peptidisc from the affinity resin . The same procedure was done in parallel , except the NSPr was omitted from the Assembly Buffer and 0 . 02% DDM was included in Buffer A , B , and C . The purified BRC complex ( 1 mg/mL ) was mixed at a 1:1 . 8 ( µg/µg ) ratio with NSPr followed by 10-fold dilution in Buffer A to decrease the LDAO concentration to 0 . 003% . For formation of low-lipid nanodiscs , the purified BRC complex was instead mixed at a 1:2 ( mol/mol ) ratio with MSP1D1 before dilution . Alternatively , an equivalent amount of BRC was diluted in Buffer A supplemented with 0 . 03% LDAO , 0 . 02% DDM , 0 . 1% SMA or 0 . 1% SDS as described . After incubation for 10 min on ice , aggregated proteins were removed by centrifugation ( 13 , 000 x g , 10 min at 4°C ) . Peptidisc formation was confirmed by analysis on CN-PAGE . Proteoliposomes were prepared at a molar protein:lipid ratio of 1:2000 . Total E . coli lipids were dissolved in chloroform , dried under nitrogen and resuspended in Buffer A + 0 . 8% β-OG . Purified MalFGK2 was added to the solubilized lipids , and the detergent was removed by overnight incubation at 4°C with Amberlite XAD-2 adsorbent beads ( Supelco ) . The proteoliposomes were isolated by ultracentrifugation ( 100 , 000 × g , 60 min at 4°C ) and resuspended in 20 mM Tris–HCl , pH 8 before use in ATPase assays . The same procedure was employed for the BRC , but a lipid mixture of DOPC:DOPG ( 80:20 mol/mol ) was utilized in place of total E . coli lipids . Equal volumes of 4% and 12% acrylamide solutions were prepared in advance ( Supplementary file 2 ) . Linear gradient gels were formed by gradual mixing of the two solutions ( 35 mL each ) at a flow rate of 2 ml/min using a 100 mL gradient mixer ( Sigma ) . The cross-linking agents , TEMED and ammonium persulfate , were added immediately before gradient mixing . Once poured , plastic wells ( Biorad ) were inserted and gels allowed to cure for 90 min before storage at 4°C . For clear-native PAGE , anode and cathode buffers consisted of Buffer N ( 37 mM Tris-HCl; 35 mM Glycine; pH 8 . 8 ) . For blue-native PAGE , anode buffer consisted of Buffer N + 180 µM Coomassie Blue G-250 , and cathode buffer contained Buffer N only . Aliquots of MalFGK2-NSPr were analyzed by static light scattering . Static light scattering analysis were performed using a WTC-050S5 column ( Wyatt Technologies ) connected to a miniDAWN light scattering detector and interferometry refractometer ( Wyatt Technologies ) . Data were recorded in real time and the molecular masses were calculated using the Debye fit method using the ASTRA software ( Wyatt Technology ) . MalFGK2-peptidisc sample ( 0 . 035 mg/mL ) reconstituted by on-column method was applied onto negatively glow-discharged carbon-coated grids ( 400 mesh , copper grid ) for 1 min , and excess liquid was removed by blotting with filter paper . Freshly prepared 1 . 5% uranyl formate ( pH 5 ) was added ( 5 µl ) for 1 min and then blotted . Around 200 digital micrographs were collected using a FEI Tecnai G2 F20 microscope operated at 200 kV and equipped with a Gatan Ultrascan 4k × 4 k Digital CCD Camera . The images were recorded at defocus between 0 . 7 and 1 . 4 µm at a magnification of 67 , 000X at the camera and a pixel size of 2 . 24 Å . Contrast transfer function parameters were determined using CTFFIND3 ( Mindell and Grigorieff , 2003 ) . We selected 31188 protein particles using e2boxer from the EMAN2 software suite ( Tang et al . , 2007 ) and extracted with a box size of 96 × 96 pixels . Particles were classified using a likelihood 2D classification with 16 seeds with the RELION-1 . 3 software suite ( Scheres , 2012 ) . A 2D variance of particles contained in side view ( 3175 particles ) was computed with SPARX to estimate the peptidisc diameter variation ( Hohn et al . , 2007 ) . The measurements were done using e2display from the EMAN2 software suite ( Corin et al . , 2011 ) on the side views shown in Figure 2C . FhuA-MSPL156 nanodiscs were prepared as previously described ( Lee et al . , 2016 ) . FhuA-NSPr was prepared by on-column peptidisc reconstitution . About 2 µg of FhuA reconstituted into either MSPL156 or NSPr was incubated with TonB23-329 ( 2 µg ) or ColM ( 5 µg ) in the presence or absence of ferricrocin for 5 min at room temperature . The protein complexes were separated by CN-PAGE and visualized by Coomassie blue staining . Neither monomeric TonB nor ColM migrate on CN-PAGE due to their isoelectric points > pH 8 . 8 . BRC peptidisc , MalFGK2 peptidisc , and FhuA peptidisc were prepared by ‘on-column’ reconstitution in 100 mM ammonium acetate , pH 7 . 0 at protein to NSPr ( g/g ) ratios of 1:1 . 8 , 1:1 . 6 , and 1:1 . 8 , respectively . Mass spectrometry measurements were performed in positive ion mode on a Synapt G2S quadrupole-ion mobility separation-time-of-flight ( Q-IMS-TOF ) mass spectrometer ( Waters , Manchester , UK ) with a nanoflow electrospray ionization ESI ( nanoESI ) source . Borosilicate capillaries ( 1 . 0 mm o . d . , 0 . 68 mm i . d . ) were pulled in-house using a P-1000 micropipette puller ( Sutter Instruments , Novato , CA ) . A voltage of ~1 . 0 kV was applied to a platinum wire was inserted into the nanoESI tip . A source temperature of 60°C and a Cone voltage of 30 V were used . Argon was used in the Trap and Transfer ion guides , at pressures of 2 . 77 × 10−2 mbar and 2 . 84 × 10−2 mbar , respectively , and the Trap and Transfer voltages were 5 V and 2 V , respectively . All data were processed using MassLynx software ( v4 . 1 ) . Spectral deconvolution was performed with the UniDec ( Marty et al . , 2015 ) deconvolution algorithm using the following parameters: m/z range – 7000 to 9500 ( MalFGK2 peptidisc ) , 5500 to 9000 ( BRC peptidisc ) , 5000 to 10000 ( FhuA peptidisc ) ; Subtract minimum - 50 . 0; Gaussian Smoothing - 10 . 0; Bin every 1 . 0; Linear m/z ( constant delta m/z ) ; Charge Range - 20 to 40 ( MalFGK2 peptidisc ) , 10 to 30 ( BRC peptidisc ) , 10 to 30 ( FhuA peptidisc ) ; Mass range - 200 , 000 to 300 , 000 ( MalFGK2 peptidisc ) , 100 , 000 to 180 , 000 ( BRC peptidisc ) , 100 , 000 to 170 , 000 ( FhuA peptidisc ) ; Sample Mass Every 1 . 0 Da; Peak FWHM ( Th ) 4 . 0; Peak Shape Function - Gaussian; Charge Smooth Window - 1 . 0; Mass Difference - 4474 . 0; Mass Smooth Window - 1 . 0; Maximum number of iterations - 1000 . Spectral files were loaded as text files containing intensity and m/z values . Absorption spectra were recorded using a Hitachi U-3010 spectrophotometer . A blank measurement was recorded in Buffer A ( +0 . 03% LDAO for detergent purified BRC ) . Samples were incubated in a PCR thermocycler at the indicated temperature , and then measured at the desired time points in a quartz cuvette at room temperature . Spectra were collected between 600 nm and 1100 nm ( scan time ∼20 s ) at intervals of 1 . 5 min . For comparisons of spectra between conditions , spectra were normalized to a value of 1 . 0 at 804 nm . The BRC complex into the indicated detergent or reconstituted into peptidiscs was incubated at varying temperatures in a PCR thermocycler for 5 min , then 3 µL of the mixture dotted onto nitrocellulose paper pre-wetted in Buffer A . The dot blot was imaged using a LICOR odyssey infrared fluorescence scanner ( excitation 680 nm , emission 700 nm ) . Fluorescence intensity was quantified by Image J . The MalFGK2 and BRC peptidiscs were prepared by on-column reconstitution on a Superdex 5/25 column equilibrated in Buffer A , followed by one additional gel filtration step to ensure full removal of free NSPr . MalFGK2 ( 1 µg ) , FhuA ( 2 µg ) , and BRC ( 2 µg ) peptidiscs were analyzed by 15% SDS-PAGE . Gels were stained with Coomassie Blue G-250 , and destained overnight before fluorescence measurement ( excitation 680 nm , emission 700 nm ) on a LICOR Odyssey scanner . The band corresponding to the NSPr peptide was quantified by densitometry using Image J and compared to a standard curve of NSPr ( 0–2 µg ) loaded on the same gel . The determined NSPr amount was then subtracted from the total amount of protein loaded on the gel to determine the amount of reconstituted membrane protein in the peptidisc . Membrane protein content in peptidisc ( g ) = total protein in peptidisc ( g ) - measured NSPr content ( g ) . We used these calculated mass measurements and the molecular weight ( MW ) for NSPr ( 4 . 5 kDa ) , MalFGK2 ( 173 kDa ) , FhuA ( 80 kDa ) and BRC ( 94 kDa ) to calculate NSPr stoichiometry as follows;NSPrStoichiometry=MWMembraneprotein ( g/mol ) MWNSPr ( g/mol ) ×MeasuredNSPrcontent ( g ) Membraneproteincontentinpeptidisc ( g ) Each experiment was repeated in triplicate on three different gels . We note that detergent-purified FhuA co-purified with a contaminant , thought to be short chain lipopolysaccharides , that migrated to the same position as NSPr , therefore FhuA was reconstituted using NSPr labelled with a biotin group ( NSPrbio ) . To quantify NSPrbio , western blots were incubated with streptavidin conjugated to Alexafluor 680 in phosphate buffered saline ( PBS ) , followed by several washes in PBS + 0 . 1% Tween . Western blots were imaged on a LICOR Odyssey scanner fluorescence ( excitation 680 nm , emission 700 nm ) , and the bands corresponding to NSPrbio quantified in Image J . The MaFGK2 and BRC peptidiscs were prepared on-bead , and the FhuA peptidisc was prepared on-column . MalFGK2 ( 40 µg ) , FhuA ( 40 µg ) , and BRC ( 80 µg ) peptidiscs were diluted to a final volume of 200 µL of Buffer A , then mixed with 800 µL of a 2:1 solution of methanol:chloroform for 10 min at 25°C in glass screw cap vials . 200 µL of chloroform and 200 µL of distilled water were added sequentially , vortexed briefly , and the resulting two phase system separated by low-speed centrifugation ( 3000 r . p . m . , 10 min ) . The organic phase was dried under nitrogen , and stored at −20°C . Total phosphate content was determined by a modified version of the malachite green assay ( Lanzetta et al . , 1979 ) . Malachite green reagent was prepared as follows: ammonium molybdate ( 4 . 2 g ) was dissolved in 100 mL of 4M HCl , then mixed with 300 mL malachite green ( 135 mg ) dissolved in distilled water . The solution was mixed for 1 hr at 4°C , filtered , and stored at 4°C before use . Dried lipid extracts were subsequently incubated with 1 mL of 70% perchloric acid for 3 hr at 130°C , and then 20 µL of the resulting solution mixed with 500 µL of the malachite green reagent for 5 min at room temperature before absorbance measurement at 660 nm . Phosphate standards ( KH2PO4 ) were diluted into perchloric acid and used to prepare a standard curve with phosphate concentrations ranging from 0 . 01 nmol to 1 nmol PO4 . For thin layer chromatography ( TLC ) analysis , dried lipids were resuspended in 30 µL of chloroform , and 10 µL were dotted onto a TLC Silica gel 60 ( Millipore ) . The TLC was developed in a solution of 35:25:3:28 chlorofrom:triethylamine:dH2O:ethanol . Plates were dried in an oven for 5 min at 150°C . Lipids were visualized by lightly wetting plates in a solution of 10% Cu2S04 in 8 . 5% phosphoric acid , followed by heating for 5 min at 150°C . The MalFGK2 ATPase activity was determined by monitoring the release of inorganic phosphate using the malachite green method ( Lanzetta et al . , 1979 ) . Protein and peptide concentrations were determined by Bradford assay ( Prehna et al . , 2012 ) . SMA polymer containing 2:1 styrene to maleic acid ratio was prepared following the procedure described by Dörr et al . ( 2014 ) . In brief , 10% of SMA 2000 ( Cray Valley ) , was refluxed for 3 hr at 80°C in 1M KOH , resulting in complete solubilization of the polymer . Polymer was then precipitated by dropwise addition of 6M HCl accompanied by stirring and pelleted by centrifugation ( 1500 x g for 5 min ) . The pellet was then washed 3 times with 50 mL of 25 mM HCl , followed by a third wash in ultrapure water and subsequent lyophilization . Lyophilized SMA was later re-suspended at 10% wt/vol in 25 mM Tris-HCl , and the pH of the solution adjusted to 8 with 1M NaOH . Peptide hydrophobic moment and electropotential was calculated using the 3D-HM calculator ( Reißer et al . , 2014 ) . Sequences corresponding to NSP or NSPr were calculated with the C-terminus specified as ( COO- ) and N-terminus specified as ( NH3+ ) . UV absorbance of solubilized peptides was measured by Nanodrop .
Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles . Here , we present the peptidisc for their facile capture into water-soluble particles . Unlike the nanodisc , which requires scaffold proteins of different lengths and precise amounts of matching lipids , reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide ( NSPr ) and no extra lipids . Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein . We demonstrate the effectiveness of this ‘one size fits all’ method using five different membrane protein assemblies ( MalFGK2 , FhuA , SecYEG , OmpF , BRC ) during ‘on-column’ , ‘in-gel’ , and ‘on-bead’ reconstitution embedded within the membrane protein purification protocol . The peptidisc method is rapid and cost-effective , and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies .
Surrounding every living cell is a biological membrane that is largely impermeable to water-soluble molecules . This hydrophobic ( or “water-hating” ) barrier preserves the contents of the cell and also regulates how the cell interacts with its environment . This latter function is critical and relies on a class of proteins that are embedded within the membrane and are also hydrophobic . The hydrophobic nature of membrane proteins is however inconvenient for biochemical studies which usually take place in water-based solutions . Therefore , membrane proteins are under-represented in biological research compared to the water-soluble ones , even though roughly one quarter of a cell’s proteins are membrane proteins . Researchers have developed a few tricks to keep membrane proteins soluble after they have been extracted from the membrane . An old but popular technique makes use of detergents , which are chemicals with opposing hydrophobic and hydrophilic properties ( hydrophilic literally means “water-loving” ) . However , even mild detergents can damage membrane proteins and will sometimes lead to experimental artifacts . More recent tricks to stabilize membrane proteins without detergents have been described but remain laborious , costly or difficult to perform . To overcome these limitations , Carlson et al . developed a simple method to stabilize membrane proteins without detergent . Called the “peptidisc” , the method uses multiple copies of a unique peptide – a short sequence of the building blocks of protein – that had been redesigned to have optimal hydrophobic and hydrophilic properties . The idea was that the peptides would wrap around the hydrophobic parts of the membrane protein , and shield them from the watery solution . Indeed , when Carlson et al . mixed this peptide with five different membrane proteins from bacteria , all were perfectly soluble and functional without detergent . The ideal ratio of peptide needed to form a peptidisc around each membrane protein was reached automatically , without having to test many different conditions . This indicates that the peptidisc acts like a “one size fits all” scaffold . The peptidisc is a new tool that will allow more researchers , including those who are not expert biochemists , to study membrane proteins . This will yield a better understanding of the structure of a cell’s membrane and how it interacts with the environment . Since the approach is both simple and easy to apply , more membrane proteins can now also be included in high-throughput searches for potential new drugs for various medical conditions .