Conformational Transitions in the Membrane Scaffold Protein of Phospholipid Bilayer Nanodiscs
2011; Elsevier BV; Volume: 10; Issue: 9 Linguagem: Inglês
10.1074/mcp.m111.010876
ISSN1535-9484
AutoresChristopher R. Morgan, Christine M. Hebling, Kasper D. Rand, Darrel W. Stafford, James W. Jorgenson, John R. Engen,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoPhospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest. Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest. Biophysical investigations of membrane proteins are notoriously difficult. A wide variety of methods have been developed to circumvent some of the difficulties, including strategies to keep membrane proteins stable and soluble in aqueous solution (1Seddon A.M. Curnow P. Booth P.J. Membrane proteins, lipids and detergents: not just a soap opera.Biochim. Biophys. Acta. 2004; 1666: 105-117Crossref PubMed Scopus (996) Google Scholar). 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Acta. 1995; 1231: 223-246Crossref PubMed Scopus (400) Google Scholar) and this has particular advantages for many protein systems, such as ion channels where compartmentalization of each side of the bilayer is required (6Morera F.J. Vargas G. González C. Rosenmann E. Latorre R. Ion-channel reconstitution.Methods Mol Biol. 2007; 400: 571-585Crossref PubMed Google Scholar). Care must be taken, however, to control the size distribution of liposomes and the stoichiometry of protein-liposome mixtures. An alternative model membrane system, termed nanodiscs, was developed in 2002 (7Bayburt T.H. Grinkova Y.V. Sligar S.G. Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins.Nano Letters. 2002; 2: 853-856Crossref Scopus (568) Google Scholar). Nanodiscs provide an environment where membrane proteins are stable and compatible with aqueous environments in a native-like phospholipid bilayer. Nanodiscs (Fig. 1) are 8–15 nm diameter phospholipid bilayer discs encircled by two copies of an amphipathic helical protein called a membrane scaffold protein (MSP) 1The abbreviations used are:apoA-1apolipoprotein A-1MSPmembrane scaffold proteinHXhydrogen exchangeHDLhigh-density lipoproteinMSP1D1membrane scaffold protein 1D1DOPC1,2-dioleoyl-sn-glycero-3-phosphocholine.. The MSPs interact with the hydrophobic tails of the lipids around the circular edge of the disc, shielding the lipid from solvent. Nanodiscs self-assemble when detergent is slowly removed from a solubilized mixture of lipid and MSP (8Denisov I.G. Grinkova Y.V. Lazarides A.A. Sligar S.G. Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.J. Am. Chem. Soc. 2004; 126: 3477-3487Crossref PubMed Scopus (790) Google Scholar, 9Shih A.Y. Arkhipov A. Freddolino P.L. Sligar S.G. Schulten K. Assembly of lipids and proteins into lipoprotein particles.J. Phys. Chem. 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A great strength of the nanodisc model membrane system is that nanodiscs make membrane proteins compatible with analytical methods that have primarily been limited to investigating soluble proteins [reviewed in: (16Borch J. Hamann T. The nanodisc: a novel tool for membrane protein studies.Biol. Chem. 2009; 390: 805-814Crossref PubMed Scopus (111) Google Scholar, 17Nath A. Atkins W.M. Sligar S.G. Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins.Biochemistry. 2007; 46: 2059-2069Crossref PubMed Scopus (357) Google Scholar)]. A wide variety of experimental techniques have been used to probe the function and structure of nanodisc-embedded membrane proteins. These techniques include, but are not limited to: surface plasmon resonance (18Glück J.M. Koenig B.W. Willbold D. Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies.Anal. Biochem. 2011; 408: 46-52Crossref PubMed Scopus (48) Google Scholar), solid-state NMR (19Kijac A. Shih A.Y. Nieuwkoop A.J. Schulten K. Sligar S.G. Rienstra C.M. Lipid-protein correlations in nanoscale phospholipid bilayers determined by solid-state nuclear magnetic resonance.Biochemistry. 2010; 49: 9190-9198Crossref PubMed Scopus (27) Google Scholar), solution NMR (15Raschle T. Hiller S. Yu T.Y. Rice A.J. Walz T. Wagner G. Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs.J. Am. Chem. Soc. 2009; 131: 17777-17779Crossref PubMed Scopus (144) Google Scholar), various single-molecule fluorescence applications (20Nath A. Trexler A.J. Koo P. Miranker A.D. Atkins W.M. Rhoades E. Single-molecule fluorescence spectroscopy using phospholipid bilayer nanodiscs.Methods Enzymol. 2010; 472: 89-117Crossref PubMed Scopus (49) Google Scholar), electron paramagnetic resonance spectroscopy (21Alvarez F.J. Orelle C. Davidson A.L. Functional reconstitution of an ABC transporter in nanodiscs for use in electron paramagnetic resonance spectroscopy.J. Am. Chem. Soc. 2010; 132: 9513-9515Crossref PubMed Scopus (61) Google Scholar), and hydrogen exchange mass spectrometry (22Hebling C.M. Morgan C.R. Stafford D.W. Jorgenson J.W. Rand K.D. Engen J.R. Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.Anal. Chem. 2010; 82: 5415-5419Crossref PubMed Scopus (116) Google Scholar). The membrane scaffold protein used in many nanodiscs is an N-terminal truncation of apolipoprotein A-1 (apoA-1), which is the primary protein component of high-density lipoprotein (HDL) particles (23Rothblat G.H. Phillips M.C. High-density lipoprotein heterogeneity and function in reverse cholesterol transport.Curr. Opin. Lipidol. 2010; 21: 229-238Crossref PubMed Scopus (271) Google Scholar). Because MSP shares many properties with apoA-1, the structural characteristics of HDL particles outline much of the functional assembly of nanodiscs. Nascent HDL particles adopt a discoidal shape when they are cholesterol poor and the incorporation and subsequent esterification of cholesterol by lecithin:cholesterol acyltransferase causes the HDL particles to mature and assume a spherical shape with three apoA-1 molecules assuming what is believed to be a trefoil conformation (24Silva R.A. Huang R. Morris J. Fang J. Gracheva E.O. Ren G. Kontush A. Jerome W.G. Rye K.A. Davidson W.S. Structure of apolipoprotein A-I in spherical high density lipoproteins of different sizes.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12176-12181Crossref PubMed Scopus (161) Google Scholar) around the newly formed sphere (25Lund-Katz S. Phillips M.C. High density lipoprotein structure-function and role in reverse cholesterol transport.Subcell. 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Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation.Biochemistry. 1997; 36: 288-300Crossref PubMed Scopus (114) Google Scholar). Although there is structural information for apoA-1 in the absence of lipids, structural details for the protein in the presence of lipids are lacking. The two crystal structures for lipid-free apoA-1 display similar (>80%) helical content whereas their tertiary structure is highly divergent. Full-length apoA-1 (28Ajees A.A. Anantharamaiah G.M. Mishra V.K. Hussain M.M. Murthy H.M. Crystal structure of human apolipoprotein A-I: insights into its protective effect against cardiovascular diseases.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2126-2131Crossref PubMed Scopus (194) Google Scholar), PDB 2A01, contains an N-terminal four-helix bundle followed by two shorter C-terminal helices. An N-terminal truncation of apoA-1 (29Borhani D.W. Rogers D.P. Engler J.A. Brouillette C.G. Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12291-12296Crossref PubMed Scopus (413) Google Scholar), PDB 1AV1, adopts a discoidal shape with four copies of apoA-1. The structure of apoA-1 in the presence of lipid is much more challenging to obtain experimentally. To date, no high resolution structures for apoA-1 in discoidal HDL particles have been solved, although several models have been proposed. The models include the picket fence (30Phillips J.C. Wriggers W. Li Z. Jonas A. Schulten K. Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks.Biophys. J. 1997; 73: 2337-2346Abstract Full Text PDF PubMed Scopus (117) Google Scholar), helical hairpin (31Tricerri M.A. Behling Agree A.K. Sanchez S.A. Bronski J. Jonas A. 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Biol. 2011; doi:10.1038/nsmb.2028Crossref Scopus (182) Google Scholar). Hydrogen exchange (HX) mass spectrometry (MS) was previously applied to discoidal HDL to try to determine the spatial arrangement of apoA-1 in discoidal HDL particles (39Wu Z. Wagner M.A. Zheng L. Parks J.S. Shy 3rd, J.M. Smith J.D. Gogonea V. Hazen S.L. The refined structure of nascent HDL reveals a key functional domain for particle maturation and dysfunction.Nat. Struct. Mol. Biol. 2007; 14: 861-868Crossref PubMed Scopus (181) Google Scholar). That work used tandem MS (MS/MS) by collision induced dissociation (CID) to attempt to measure deuterium at single residue resolution and resulted in a solar flares model for discoidal HDL. Localizing deuterium by means of CID has been shown to be nearly impossible because of deuterium scrambling in the gas phase; MS/MS of deuterated peptides requires much less internal excitation, such as afforded by electron transfer dissociation, to avoid scrambling (40Rand K.D. Zehl M. Jensen O.N. Jørgensen T.J. Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry.Anal. Chem. 2009; 81: 5577-5584Crossref PubMed Scopus (180) Google Scholar, 41Zehl M. Rand K.D. Jensen O.N. Jørgensen T.J. Electron transfer dissociation facilitates the measurement of deuterium incorporation into selectively labeled peptides with single residue resolution.J. Am. Chem. Soc. 2008; 130: 17453-17459Crossref PubMed Scopus (139) Google Scholar). The solar flares model derived from CID-assisted HX MS was later shown to be inaccurate (42Shih A.Y. Sligar S.G. Schulten K. Molecular models need to be tested: the case of a solar flares discoidal HDL model.Biophys. J. 2008; 94: L87-89Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and the exact same data were reused (43Gogonea V. Wu Z. Lee X. Pipich V. Li X.M. Ioffe A.I. Didonato J.A. Hazen S.L. Congruency between biophysical data from multiple platforms and molecular dynamics simulation of the double-super helix model of nascent high-density lipoprotein.Biochemistry. 2010; 49: 7323-7343Crossref PubMed Scopus (34) Google Scholar) to support a different model, the proposed double superhelical model (44Wu Z. Gogonea V. Lee X. Wagner M.A. Li X.M. Huang Y. Undurti A. May R.P. Haertlein M. Moulin M. Gutsche I. Zaccai G. Didonato J.A. Hazen S.L. Double superhelix model of high density lipoprotein.J. Biol. Chem. 2009; 284: 36605-36619Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This model has an elliptical cross section and is the first to deviate from the widely accepted discoidal shape; however the validity of this model remains controversial (45Jones M.K. Zhang L. Catte A. Li L. Oda M.N. Ren G. Segrest J.P. Assessment of the validity of the double superhelix model for reconstituted high density lipoproteins: a combined computational-experimental approach.J. Biol. Chem. 2010; 285: 41161-41171Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In terms of nanodiscs, as opposed to HDL particles, small-angle neutron and x-ray scattering (46Skar-Gislinge N. Simonsen J.B. Mortensen K. Feidenhans'l R. Sligar S.G. Lindberg Møller B. Bjørnholm T. Arleth L. Elliptical structure of phospholipid bilayer nanodiscs encapsulated by scaffold proteins: casting the roles of the lipids and the protein.J. Am. Chem. Soc. 2010; 132: 13713-13722Crossref PubMed Scopus (100) Google Scholar), solid-state NMR spectroscopy (47Li Y. Kijac A.Z. Sligar S.G. Rienstra C.M. Structural analysis of nanoscale self-assembled discoidal lipid bilayers by solid-state NMR spectroscopy.Biophys. J. 2006; 91: 3819-3828Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and molecular modeling (48Shih A.Y. Denisov I.G. Phillips J.C. Sligar S.G. Schulten K. Molecular dynamics simulations of discoidal bilayers assembled from truncated human lipoproteins.Biophys. J. 2005; 88: 548-556Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) of nanodiscs generally support a double-belt model, similar to that of discoidal HDL particles. An example of the double-belt model structure of a nanodisc is shown in Fig. 1 where the two copies of apoA-1 encircle the lipid disc in an antiparallel manner and the two copies are rotated such that the maximum number of intermolecular salt-bridges are formed. When apoA-1 is wrapped around the perimeter of the lipid disc it is primarily helical in nature (the 10 amphipathic helices are highlighted in Fig. 1B). Because the conformation of lipid-free apoA-1 differs dramatically from that of lipid-associated apoA-1, it is reasonable to assume that lipid-free MSP also has a different conformation relative to lipid-associated MSP. To test this hypothesis, and to provide other vital data about the MSP, as described below, we examined the hydrogen exchange of lipid-free MSP and MSP in assembled nanodiscs. We previously reported on HX MS methods to analyze the conformation of a transmembrane protein embedded in a nanodisc (22Hebling C.M. Morgan C.R. Stafford D.W. Jorgenson J.W. Rand K.D. Engen J.R. Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.Anal. Chem. 2010; 82: 5415-5419Crossref PubMed Scopus (116) Google Scholar). In that work, a modified HX MS protocol was employed to enhance nanodisc disassembly and digestion as well as remove phospholipids prior to MS analysis. Although MSP was present in those experiments and deuterium exchange occurred in MSP, the MSP itself was not characterized. In the present work, both lipid-free MSP and MSP in fully assembled nanodiscs were studied and compared. The MSP [specifically MSP1D1, see (8Denisov I.G. Grinkova Y.V. Lazarides A.A. Sligar S.G. Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.J. Am. Chem. Soc. 2004; 126: 3477-3487Crossref PubMed Scopus (790) Google Scholar)] was analyzed at a concentration of <5 μm where lipid-free MSP1D1 was monomeric and the nanodiscs were monodisperse. The results reveal a gross structural rearrangement and decreased deuteration of MSP1D1 upon lipid-association. However, MSP1D1 in nanodiscs remained dynamic and still became significantly deuterated even though the models indicate a high percentage of helical content and hydrophobic interactions with the lipids. EX1 kinetic signatures indicative of cooperative unfolding were observed in several regions of MSP in both the lipid-free and lipid-associated states. Our results provide important information for understanding MSP1D1 conformation in nanodiscs, demonstrate the feasibility of studying biologically relevant HDL particles by this method, offer essential control data that characterize and validate proper nanodisc assembly, and catalogue the peptides and results expected for the MSP1D1 background signals that will be present in any HX MS experiment involving nanodiscs to study peripheral or integral membrane proteins associated with nanodiscs. MSP1D1 expression and purification was carried out as previously described (8Denisov I.G. Grinkova Y.V. Lazarides A.A. Sligar S.G. Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.J. Am. Chem. Soc. 2004; 126: 3477-3487Crossref PubMed Scopus (790) Google Scholar). In short, the MSP1D1 plasmid was obtained from Add Gene and expressed in Escherichia coli BL21 Codon Plus (DE3) cells. For purification, the protein was isolated by nickel affinity chromatography and purity was confirmed by polyacrylamide gel electrophoresis and electrospray mass spectrometry. Fractions containing MSP1D1 were pooled and dialyzed against 20 mm Tris/HCl pH 7.4, 0.1 m NaCl, 0.5 mm EDTA, and 0.02% NaN3. Protein concentration was determined by absorbance at 280 nm using ε = 21000 cm−1M−1. Nanodisc self-assembly was carried out as previously described (8Denisov I.G. Grinkova Y.V. Lazarides A.A. Sligar S.G. Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.J. Am. Chem. Soc. 2004; 126: 3477-3487Crossref PubMed Scopus (790) Google Scholar). Purified MSP1D1 was added to a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/deoxycholate solubilized mixture (2:1) to a ratio of 67:1 DOPC:MSP1D1 and incubated for one hour at 4 °C. After incubation, detergent was removed during a 2 h gentle rotation with damp Biobeads SM-2 (BioRad) at 4 °C. The disk preparations were purified by size exclusion chromatography on a Tosoh Biosciences TSKGEl BioAssist G3SWXL 7.8 × 300 mm (5 μm, 250 Å) column run with 50 mm Tris/HCl pH 7.0, 0.15 m NaCl, 0.02% NaN3 at 0.5 ml/minute. Fractions containing purified nanodiscs were isolated and concentrated by Millipore Microcon YM-30 centrifugal filters. HX MS experiments were similar to those described previously (22Hebling C.M. Morgan C.R. Stafford D.W. Jorgenson J.W. Rand K.D. Engen J.R. Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.Anal. Chem. 2010; 82: 5415-5419Crossref PubMed Scopus (116) Google Scholar). Both lipid-free and lipid-associated MSP1D1 were labeled with deuterium in an identical fashion to ensure that both the exchange reaction and quench conditions were the same for both conformational states. Continuous labeling exchange experiments were initiated by diluting 25 μl of sample solution (4 μm lipid-free MSP1D1 or 4 μm MSP1D1 in nanodiscs) 10-fold in 99% deuterium oxide buffer (50 mm Tris/HCl, 0.15 m NaCl, 0.02% NaN3, 2H2O, pD 7.0) at 21 °C. At predetermined time points (ranging from 1 s to 4 h) the labeling reaction was quenched to pH 2.5 by the addition of 0.68 μl of concentrated formic acid (using a 2-μl pipet with filter tip) and placed on ice. All labeling experiments were prepared by hand with pipets, including the short time points of 1 and 5 s (with practice, reproducible labeling and quenching can be accomplished with two manual pipets for a 1 s labeling time point). To retain as much of the deuterium label as possible, all solutions after the quenching step were maintained at pH 2.5 and 0–4 °C before analysis by UPLC/MS. Immediately after quenching, ice-cold sodium cholate solution (10 mm sodium cholate in H2O) was added to a final 25:1 cholate:DOPC molar ratio. POROS 20AL beads [6 μl as a 50% slurry in H2O, 0.08% TFA, pH 2.5, (Applied Biosystems, Foster City, CA)] containing covalently coupled porcine pepsin (49Wang L. Pan H. Smith D.L. Hydrogen exchange-mass spectrometry: optimization of digestion conditions.Mol. Cell Proteomics. 2002; 1: 132-138Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) were then added and the mixture was incubated on ice. After 4 mins of digestion, 3 mg of zirconium oxide coated silica particles (Supelco HybridSPE, #55261-U) were added to the digestion mixture to precipitate phospholipid. After 1 min of incubation with the ZrO2 particles the mixture was spin filtered (0.45 μm cellulose acetate filter, prechilled) at 4 °C for 1 min to remove the pepsin and ZrO2 beads. Flow through from the spin filtration was immediately injected into a Waters nanoAcquity UPLC with hydrogen exchange technology (50Wales T.E. Fadgen K.E. Gerhardt G.C. Engen J.R. High-speed and high-resolution UPLC separation at zero degrees Celsius.Anal. Chem. 2008; 80: 6815-6820Crossref PubMed Scopus (250) Google Scholar) in which the valves, sample loop, trap and analytical columns were all maintained at 0 °C. The regular online pepsin column normally used in this system was not present and was instead replaced by a union. Upon injection, the peptic peptides were trapped on a pre-column (Waters VanGuard C18, 1.7 μm, 2.1 × 5 mm) (Milford, MA) and desalted with 0.05% formic acid in H2O, pH 2.5, 100 μl/minute for 5 min. Peptides were eluted from the trap to the analytical column (Waters XBridge C18, 1.7 μm, 1.0 mm × 100 mm). A second pre-column (identical to the above one) was placed before the analytical column to prevent any lipid that had not been precipitated from entering the analytical column. Peptides were separated with an 8–40% gradient of 0.05% formic acid in acetonitrile (pH 2.5) over 6 min at a flow rate of 40 μl/min. Throughout an entire experiment, the organic phase never exceeded 85% to ensure that any lipid captured on the trap or guard column did not elute into the mass spectrometer. After a complete HX MS time course analysis, the column and traps were regenerated by flowing 95% acetonitrile through the system. Note that the
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