Oligomeric State of the Escherichia coli Metal Transporter YiiP
2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês
10.1074/jbc.m407044200
ISSN1083-351X
Autores Tópico(s)Electrochemical Analysis and Applications
ResumoYiiP is a 32.9-kDa metal transporter found in the plasma membrane of Escherichia coli (Chao, Y., and Fu, D. (2004) J. Biol. Chem. 279, 17173–17180). Here we report the determination of the YiiP oligomeric state in detergent-lipid micelles and in membranes. Molecular masses of YiiP solubilized with dodecyl-, undecyl-, decyl-, or nonyl-β-d-maltoside were measured directly using size-exclusion chromatography coupled with laser light-scattering photometry, yielding a mass distribution of YiiP homo-oligomers within a narrow range (68.0–68.8 kDa) that equals the predicted mass of a YiiP dimer within experimental error. The detergent-lipid masses associated with YiiP in the mixed micelles were found to increase from 135.5 to 232.6 kDa, with an apparent correlation with the alkyl chain length of the maltoside detergents. Cross-linking the detergent-solubilized YiiP with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) resulted in a dimeric cross-linked product in an EDC concentration-dependent manner. The oligomeric state of the purified YiiP in reconstituted membranes was determined by electron microscopic analysis of two-dimensional YiiP crystals in negative stain. A projection structure calculated from measurable optical diffractions to 25 Å revealed a pseudo-2-fold symmetry within a molecular boundary of ∼75 × 40 Å, indicative of the presence of YiiP dimers in membranes. These data provide direct structural evidence for a dimeric association of YiiP both in detergent-lipid micelles and in the reconstituted lipid bilayer. The functional relevance of the dimeric association in YiiP is discussed. YiiP is a 32.9-kDa metal transporter found in the plasma membrane of Escherichia coli (Chao, Y., and Fu, D. (2004) J. Biol. Chem. 279, 17173–17180). Here we report the determination of the YiiP oligomeric state in detergent-lipid micelles and in membranes. Molecular masses of YiiP solubilized with dodecyl-, undecyl-, decyl-, or nonyl-β-d-maltoside were measured directly using size-exclusion chromatography coupled with laser light-scattering photometry, yielding a mass distribution of YiiP homo-oligomers within a narrow range (68.0–68.8 kDa) that equals the predicted mass of a YiiP dimer within experimental error. The detergent-lipid masses associated with YiiP in the mixed micelles were found to increase from 135.5 to 232.6 kDa, with an apparent correlation with the alkyl chain length of the maltoside detergents. Cross-linking the detergent-solubilized YiiP with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) resulted in a dimeric cross-linked product in an EDC concentration-dependent manner. The oligomeric state of the purified YiiP in reconstituted membranes was determined by electron microscopic analysis of two-dimensional YiiP crystals in negative stain. A projection structure calculated from measurable optical diffractions to 25 Å revealed a pseudo-2-fold symmetry within a molecular boundary of ∼75 × 40 Å, indicative of the presence of YiiP dimers in membranes. These data provide direct structural evidence for a dimeric association of YiiP both in detergent-lipid micelles and in the reconstituted lipid bilayer. The functional relevance of the dimeric association in YiiP is discussed. YiiP is a member of the cation diffusion facilitator (CDF) 1The abbreviations used are: CDF, cation diffusion facilitator; SEC, size-exclusion chromatography; EDC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; ABC, ATP binding cassette; LS, light scattering; UV, ultraviolet; RI, refractive index; DDM, n-dodecyl-β-dmaltoside; UDM, undecyl-β-d-maltoside; DM, decyl-β-d-maltoside; NM, nonyl-β-d-maltoside; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; HPLC, high performance liquid chromatography; TECP, Tris(2-carboxyethyl) phosphine hydrochloride; MES, 4-morpholineethanesulfonic acid. family that functions principally as a metal transporter (1Chao Y. Fu D. J. Biol. Chem. 2004; 279: 17173-17180Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 2Chao Y. Fu D. J. Biol. Chem. 2004; 279: 12043-12050Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Analysis of genome sequences shows that CDFs represent a ubiquitous protein family, encompassing more than 400 evolutionarily related members found from bacteria and yeast to plants and mammals (3Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: 138-141Crossref PubMed Google Scholar). This protein family is characterized by an N-terminal hydrophobic domain followed by a C-terminal hydrophilic region that is highly variable both in sequence and in length (4Paulsen I.T. Saier Jr., M.H. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (295) Google Scholar). Despite large variability in the hydrophilic region, all CDF family members identified so far, prokaryotic or eukaryotic, are predominantly involved in controls of cytosolic zinc buildup during zinc excesses, either by facilitating zinc efflux from cytosol to the outside of cells or by transporting the cytosolic zinc into intracellular organelles (5Kambe T. Yamaguchi-Iwai Y. Sasaki R. Nagao M. Cell. Mol. Life Sci. 2004; 61: 49-68Crossref PubMed Scopus (338) Google Scholar). This zinc-transporting function is attributed to the homologous hydrophobic domain, which is thought to be composed of a bundle of six transmembrane segments in an α-helical configuration, a structural theme found in many other membrane channels and transporters, including those in the aquaporin family (6Agre P. Bonhivers M. Borgnia M.J. J. Biol. Chem. 1998; 273: 14659-14662Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar) and those within the ATP binding cassette (ABC) transporter superfamily (7Tomii K. Kanehisa M. Genome Res. 1998; 8: 1048-1059Crossref PubMed Scopus (106) Google Scholar). The order of subunit oligomerization is an important structural parameter in connection with subunit arrangements and mechanisms of transmembrane activities. Crystal structures of aquaporins revealed a tetrameric structure with four independent transmembrane channels located in the center of each monomer (8Fu D. Libson A. Miercke L.J. Weitzman C. Nollert P. Krucinski J. Stroud R.M. Science. 2000; 290: 481-486Crossref PubMed Scopus (886) Google Scholar), whereas an ABC transporter MsbA was found to be a dimer with a substrate translocation pathway along the dimeric interface (9Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (585) Google Scholar). Different oligomeric structures of these membrane proteins illustrate how transmembrane active sites may evolve differently from a common six-spanner architecture. Consequently, subunit oligomerization may contribute to the molecular architecture of transmembrane channels and translocation pathways that constitute the structural basis for distinctive transmembrane functions; aquaporins are selective pores that allow transmembrane permeation of water and glycerol by simple diffusion (10Borgnia M.J. Agre P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2888-2893Crossref PubMed Scopus (185) Google Scholar), whereas ABC transporters actively move diverse substrates across the membrane at the expense of adenosine triphosphate hydrolysis (11Hyde S.C. Emsley P. Hartshorn M.J. Mimmack M.M. Gileadi U. Pearce S.R. Gallagher M.P. Gill D.R. Hubbard R.E. Higgins C.F. Nature. 1990; 346: 362-365Crossref PubMed Scopus (957) Google Scholar). CDFs operate with an antiport mechanism that utilizes the potential energy in the form of a transmembrane proton gradient to drive the movement of zinc in a reverse direction (2Chao Y. Fu D. J. Biol. Chem. 2004; 279: 12043-12050Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 12Lee S.M. Grass G. Haney C.J. Fan B. Rosen B.P. Anton A. Nies D.H. Rensing C. FEMS Microbiol. Lett. 2002; 215: 273-278Crossref PubMed Google Scholar). The oligomeric state of CDF transporters has not yet been established, and little is known about the structure of any CDF besides the fact that most CDFs are predicted to be six-spanning membrane proteins (4Paulsen I.T. Saier Jr., M.H. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (295) Google Scholar). Therefore, determining the quaternary structure of a CDF transporter is paradigmatic for understanding the structural basis of zinc transport. Two Escherichia coli CDF proteins, YiiP and ZitB, provide unique models for the study of CDFs at the molecular level. The N-terminal hydrophobic domain of YiiP contains ∼200 amino acids followed by a C-terminal hydrophilic region of ∼100 amino acids. Chromosomal expression of both E. coli CDFs was found to be inducible by zinc in a concentration-dependent manner, suggesting that both CDFs are involved in zinc homeostatic controls against environmental zinc fluctuation (13Grass G. Fan B. Rosen B.P. Franke S. Nies D.H. Rensing C. J. Bacteriol. 2001; 183: 4664-4667Crossref PubMed Scopus (125) Google Scholar). YiiP and ZitB were overexpressed, solubilized by non-ionic detergents, and purified to homogeneity in mixed micelles with detergents and lipids. The purified protein, which appeared to be in a native-like state, was reconstituted into the lipid bilayer and subjected to stopped-flow kinetic analysis (2Chao Y. Fu D. J. Biol. Chem. 2004; 279: 12043-12050Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The transport of zinc through ZitB was found to be a two-step kinetic process involving an equilibrium metal binding followed by a proton-linked transport that moves the bound metal ion across the membrane. As such, an inwardly orientated proton gradient across the plasma membrane of E. coli serves as the driving force for the efflux of the cytoplasmic zinc. Further mechanistic studies by isothermal titration calorimetry with purified YiiP revealed a mutually competitive binding site common to Zn2+, Cd2+, and Hg2+ and a set of non-competitive binding sites (1Chao Y. Fu D. J. Biol. Chem. 2004; 279: 17173-17180Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In the present study we sought to determine the oligomeric state of YiiP in an effort to gain structural insights into zinc transport by CDFs. The oligomeric state of YiiP could be derived from molecular mass measurements (14Wyatt P. Anal. Chim. Acta. 1993; 272: 1-40Crossref Scopus (1298) Google Scholar). Previous studies with analytical SEC showed that the purified YiiP was eluted as a major monodisperse species with a retention time corresponding to an apparent molecular mass of 190 kDa (2Chao Y. Fu D. J. Biol. Chem. 2004; 279: 12043-12050Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Any oligomeric state of YiiP from a dimeric to a tetrameric form could account for this apparent molecular mass because the retention time, a measure of protein Stoke's radius and geometry, could vary considerably depending on the amount of detergents and lipids bound to YiiP. To determine definitively the molecular mass of YiiP oligomers in mixed micelles, we used an absolute mass analysis method based on size-exclusion HPLC in conjunction with simultaneous detections of ultraviolet absorption (UV), laser light-scattering (LS), and differential refractive index (RI) of the column effluent (15Hayashi Y. Matsui H. Takagi T. Methods Enzymol. 1989; 172: 514-528Crossref PubMed Scopus (98) Google Scholar). This UV-LS-RI method yielded molecular mass estimates for YiiP oligomers in mixed micelles irrespective of the size, shape, and lipid-detergent constituent of the mixed micelles. Another advantage of this light-scattering analysis method is its applicability to unstable membrane proteins that tend to denature rapidly in detergent micelles. HPLC runs and molecular mass calculations can be completed within 30 min, representing a clear improvement over the analytical ultracentrifugation method that requires several days to attain sedimentation equilibrium (16Reynolds J.A. Tanford C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4467-4470Crossref PubMed Scopus (112) Google Scholar). Here we describe the molecular mass analysis of YiiP oligomers in mixed micelles with endogenous lipids and four maltoside detergents. The protein masses found were approximately equal to the expected molecular mass of a YiiP dimer. The detergent-lipid masses associated with the YiiP dimer in the mixed micelles varied according to the type of the maltoside detergent. To further characterize the oligomeric state of YiiP in mixed micelles, chemical cross-linking was employed using a zero-spacer cross-linker EDC (17Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Crossref PubMed Scopus (726) Google Scholar). Because the subunit organization in the membrane environment may be different from that in the detergent-solubilized form, the quaternary structure of YiiP was also studied for the membrane-embedded state by two-dimensional crystallization of YiiP and electron microscopic imaging (18Werten P.J. Remigy H.W. de Groot B.L. Fotiadis D. Philippsen A. Stahlberg H. Grubmuller H. Engel A. FEBS Lett. 2002; 529: 65-72Crossref PubMed Scopus (67) Google Scholar). Our results indicate that YiiP is a dimer both in detergent-lipid micelles and in membranes. Overexpression and Purification of YiiP—YiiP was overexpressed and purified as described (1Chao Y. Fu D. J. Biol. Chem. 2004; 279: 17173-17180Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Briefly, a frozen stock of BL21(DE3)pLysS cells (Novagen Inc., Madison, WI) containing pYiiP-TB-His plasmids was inoculated into an auto-inducing medium for unattended induction of protein overexpression. The overnight culture was harvested, and membrane vesicles were prepared and then subjected to detergent solubilization using n-dodecyl-β-d-maltoside (DDM, Anatrace, Maumee, OH). The detergent-solubilized YiiP-TB-His was purified by nickel affinity chromatography followed by a desalting step to remove imidazole that was present in the eluate of the Ni2+-nitrilotriacetic acid superflow column (Qiagen, Valencia, CA). Next the His tag was removed by overnight thrombin digestion, and the resultant YiiP was further purified by size-exclusion HPLC using a TSK 3000SWXL column (TosoHaas, Montgomeryville, PA) equilibrated with the following mobile phase: 20 mm HEPES, pH 7.0, 100 mm NaCl, 12.5% glycerol, 0.05% DDM, and 0.25 mm Tris(2-carboxyethyl) phosphine hydrochloride (TECP)). The column effluent was monitored by a UV detector (Beckman Coulter, Fullerton, CA), and the purified YiiP was collected as a discrete chromatographic fraction using a Beckman SC100 fraction collector. Sample Preparation for Light-scattering Experiments—All protein samples for light-scattering experiments were adjusted to ∼1.0 mg/ml, dialyzed against the HPLC mobile phase at 10 °C using a 100-kDa molecular mass cutoff DispoDialyzer (Spectrum Laboratories, Rancho Dominguez, CA), and then centrifuged at 140,000 × g for 30 min to remove any precipitate. The mobile phase was also filtered with a 0.22-μm Millipore GS filter and degassed thoroughly. For light-scattering experiments with different detergents, protein samples were dialyzed with frequent changes of the dialysis bulk solution containing one of the following four detergents as indicated: 0.05% DDM, 0.06% undecyl-maltoside (UDM), 0.15% decyl-maltoside (DM) or 0.4% nonyl-maltoside (NM). For light-scattering experiments with YiiP delipidated to varied levels, samples were prepared by passing YiiP through the TSK column successively and collecting the eluted YiiP peak at each passage. Size-exclusion Chromatography and Multiangle Laser Light-scattering Measurement—The instrument setup used for SEC-light scattering experiments consisted of a System-Gold HPLC system (Beckman Coulter) connected in series with a DAWN DSP light-scattering detector (Wyatt Technology) and an OPTILAB DSP RI interferometric refractometer detector (Wyatt Technology). Analytical size-exclusion chromatography was performed at 20 °C using an TSK 3000SWXL column (TosoHaas) equilibrated with a mobile phase containing 100 mm NaCl, 20 mm HEPES, pH 7.0, 0.25 mm TECP and one of the follow detergents as indicated: 0.05% DDM, 0.06% UDM, 0.15% DM, or 0.4% NM. 100 μl of purified YiiP sample at ∼1.0 mg/ml was injected into the column and eluted at a flow rate of 0.5 ml/min. The column effluent was monitored in-line with three detectors that simultaneously monitored UV absorption, light scattering, and refractive index, respectively. The resultant three chromatograms were aligned with that of the LS output after corrections for the interdetector volume delays between the UV-to-LS and RI-to-LS detector. Detector outputs were digitized and acquired by a Pentium workstation running Astra software (Wyatt Technology). Calculation of the Protein Core Mass in Protein-Detergent-Lipid Micelles—It has been established that the angular dependence of scattered light can be ignored for globular proteins with molecular masses smaller than 5000 kDa (19Takagi T. J. Chromatogr. 1990; 506: 409-416Crossref Scopus (67) Google Scholar). The molecular mass of YiiP-detergent-lipid micelles was estimated to be 190 kDa by analytical size-exclusion chromatography. The corresponding SEC light-scattering data, collected simultaneously at 16 different angles between 14 and 163°, remained unchanged within experimental error. Thus, the scattering light signal measured at 90° was used in approximation for all calculations. The second virial coefficient term in the protein mass calculation can also be ignored at protein concentrations encountered in the chromatographic analysis (20Wen J. Arakawa T. Philo J.S. Anal. Biochem. 1996; 240: 155-166Crossref PubMed Scopus (443) Google Scholar). Based on these two approximations, the molecular mass of YiiP (Mp) in protein-detergent-lipid micelles can be determined by a three-detector method (15Hayashi Y. Matsui H. Takagi T. Methods Enzymol. 1989; 172: 514-528Crossref PubMed Scopus (98) Google Scholar) using the equation, Mp=k1(output)LS(output)UVA(output)RI2(Eq. 1) where k1 is an instrument response factor, (output)LS, (output)UV and (output)RI are elution peak intensities, obtained in the unit of volt by the LS, UV, and RI detector, and A is the extinction coefficient of the protein. Calculation of the Detergent-Lipid Mass in Protein-Detergent-Lipid Micelles—The amount of detergent and lipid bound to the solubilized YiiP can be calculated according to the value of differential refractive index (dnc/dcp), which is defined as the ratio of the change in refractive index of the protein-detergent-lipid complex to the change of the protein concentration. Thus, (dnc/dcP)=k2A(output)RI(output)UV(Eq. 2) where k2 is an instrument response factor (15Hayashi Y. Matsui H. Takagi T. Methods Enzymol. 1989; 172: 514-528Crossref PubMed Scopus (98) Google Scholar). The value of dnc/dcp is the sum of weighted contributions of three components in the protein-detergent-lipid ternary complex (15Hayashi Y. Matsui H. Takagi T. Methods Enzymol. 1989; 172: 514-528Crossref PubMed Scopus (98) Google Scholar). The differential refractive index of YiiP (dnp/dcp) is assumed to be 0.187 ml/g, a constant known for all regular proteins (15Hayashi Y. Matsui H. Takagi T. Methods Enzymol. 1989; 172: 514-528Crossref PubMed Scopus (98) Google Scholar). The differential refractive indexes of detergents (dnd/dcd) and lipids (dnl/dcl) were determined experimentally. Therefore, the weight ratios of the bound detergents δd and lipids δl (g/g of YiiP) in the YiiP-detergent-lipid micelles can be derived according to Equation 3. (dnc/dcP)=0.187+δd(dnd/dcd)+δl(dnl/dcl)(Eq. 3) Measurements of the Extinction Coefficient A and Instrument Response Factors k1 and k2—Protein concentrations in the detergent solution were estimated by the BCA assay (Pierce). The UV absorption at 280 nm of the identical protein sample was measured, and the extinction coefficient A was calculated as the ratio of the optical density at 280 nm to the value obtained by the BCA assay. k1 and k2 values were determined by calibrating detector outputs using Equation 1 or 2 to match the Mp or dnc/dcp value with the respective value of GlpF, a tetrameric integral membrane protein calibrator that can be purified as a monodisperse species in the identical detergent condition used for light-scattering analysis of YiiP. Briefly, 100 μl of purified GlpF at 1 mg/ml was injected into the size-exclusion column, and the outputs of UV, LS, and RI detector were recorded. k1 was calculated based on Equation 1 using the theoretical molecular mass for GlpF tetramer (116 kDa) and an extinction coefficient A of 1.74 ml/mg. The k2 value was obtained according to Equation 2, where the dnc/dcp value for GlpF was determined off-line using the OPTILAB DSP RI detector operating in a batch mode. The refractive index nc of the GlpF-detergent-lipid micellar complex was recorded at five different GlpF concentrations. The value for dnc/dcp was derived from the slope of the plot: nc versus the GlpF concentration. Mass Spectrometric Analysis—A α-cyano-4-hydroxycinnamic acid matrix was prepared as a saturated solution in a 2:1 mixture of acetonitrile and water. Aliquots of protein in a series of 1:2 dilutions were mixed with the matrix solution and then spotted onto a sample plate using the sandwich method (21Beavis R. Chait B. Methods Enzymol. 1996; 70: 519-551Crossref Google Scholar). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometric data were collected using a Voyager Biospectrotometry Work station, operated in linear mode. EDC Cross-linking—The HPLC-purified YiiP samples were dialyzed overnight to equilibrate with the following EDC reaction buffer: 100 mm MES, pH 5.5, 100 mm NaCl, 0.1 mm CdSO4, 0.05% DDM, 20% glycerol, and 0.25 mm TECP. Cross-linking reactions were initiated by adding EDC to 1.0 mg/ml YiiP to a final concentration as indicated. The reaction mixtures were incubated at 25 °C for 2 h, and then β-mercaptoethanol was added to 100 mm to terminate the reactions. The resultant reaction mixtures were centrifuged at 14,000 × g for 1 h, and then supernatants were injected to an TSK column that was equilibrated and eluted with 20 mm HEPES, pH 7.0, 100 mm NaCl, 0.05% DDM, 12.5% glycerol, and 0.25 mm TECP. Column effluents were monitored by UV absorption, and peak fractions were collected at a retention time corresponding to the peak maximum of the identical YiiP sample before EDC cross-linking. The collected peak fractions were subjected to SDS-PAGE analyses in the presence of 2 mm β-mercaptoethanol and stained with Coomassie Fluor Orange (Molecular Probes) for protein visualization. Two-dimensional Crystallization—The purified YiiP (∼1 mg/ml) was reconstituted with E. coli polar lipids (Avanti Polar Lipids, Inc.) at various lipid-to-protein ratios ranging from 0.5 to 2.0 (w/w). Lipids were solubilized in 0.05% UDM in a 20 mm HEPES buffer, pH 7.0, containing 100 mm NaCl, 5 mm MgCl2, 0.02% NaN3, and 0.1 mm 0.25 mm TECP. The detergent was slowly removed by dialysis against a bulk pH buffer in a controlled manner over 3–5 days (22Kuhlbrandt W. Q. Rev. Biophys. 1988; 21: 429-477Crossref PubMed Scopus (136) Google Scholar). Two-dimensional crystals of YiiP with a size ranging from 0.5 to 1 μm were obtained at a lipid-to-protein ratio between 0.5 and 1.0. Electron Microscopy and Image Analysis—A 5-μl droplet of the two-dimensional crystal solution was deposited onto a glow-discharged 300-mesh copper grid covered with a thin layer of continuous carbon film. After a 1-min incubation, excess solution on the grid was blotted with a piece of filter paper, and the grid was washed with 2 drops of deionized water, stained with a 2% uranyl acetate aqueous solution for 30s, and then blotted and left to air-dry. Electron microscopy was performed with a Jeol-1200EX microscope, and images were recorded on a Gatan 791 CCD camera. Standard image processing (23Amos L.A. Henderson R. Unwin P.N. Prog. Biophys. Mol. Biol. 1982; 39: 183-231Crossref PubMed Scopus (409) Google Scholar), including determining image defocus values, Fourier transform, indexing reflections, and unbending lattice distortions, were performed on an SGI Fuel work station with the software packages of MRC image2000 (24Crowther R.A. Henderson R. Smith J.M. J. Struct. Biol. 1996; 116: 9-16Crossref PubMed Scopus (665) Google Scholar) and Spider (25Frank J. Radermacher M. Penczek P. Zhu J. Li Y. Ladjadj M. Leith A. J. Struct. Biol. 1996; 116: 190-199Crossref PubMed Scopus (1808) Google Scholar). Calibration of the UV-LS-RI System—The instrument response factor k1 was determined by calibrating the outputs of UV, LS, and RI detector according to Equation 1 using GlpF, a 29-kDa aquaporin that was solubilized and purified following the identical procedure employed for the solubilization and purification of YiiP. Size-exclusion HPLC analysis of GlpF revealed a major peak (P1) with a retention time at 15.5 min followed by a minor peak (P2) at 19.5 min (Fig. 1A). It appeared that the LS and RI detector had more pronounced responses to the minor peak; particularly, the height of the RI output at P2 rose to ∼80% of the height at P1. In a set of control experiments, injections of detergent-lipid mixtures only caused base line-level UV responses but elicited significant LS and RI responses in a concentration-dependent manner. It is known that the RI response reflects the concentration of all solutes, including those with and without chromophores, whereas UV absorption is only derived from the UV absorptivity of chromophore-containing species. The negligible UV response to P2 fraction was indicative of a non-protein nature of the P2 fraction, likely attributed to empty detergent-lipid micelles. On the other hand, the P1 fraction with its dual UV and RI responses corresponded to GlpF in mixed micelles with detergents and bound lipids that may have survived the purification process. Therefore, system calibration was carried out using outputs of UV, LS, and RI detectors across the P1 maxima. A k1 value of 115.2 was obtained that allowed the outputs of UV, LS, and RI to be converted to the theoretical value of the GlpF tetrameric molar mass (116 kDa). The instrument response factor k2 was calculated based on an experimentally measured dnc/dcp value of GlpF. Off-line measurements of nc showed a series of plateaus when back-flushing the RI detector with five evenly spaced GlpF dilutions from a 2.4 mg/ml stock solution (Fig. 1B). The RI output collected from each concentration plateau was plotted against the GlpF concentration, producing a value of 0.49 ml/g for dnc/dcp (Fig. 1B, inset). Using this measured dnc/dcp value, a k2 value of 0.13 was obtained that allowed conversion of RI and UV outputs to the actual dnc/dcp value according to Equation 2. Molecular Mass of YiiP Oligomers in Mixed Micelles—YiiP-TB-His was overexpressed, solubilized with DDM, and purified by metal affinity chromatography in the form of (YiiP-TB-His)-DDM-lipid micelles. The His tag was cleaved by thrombin digestion at a cleavage recognition sequence LVPR↓GS that was inserted between the His tag and the C-terminal sequence of YiiP, resulting in a tag-free YiiP variant with a predicted molecular mass of 32.6 kDa. Size-exclusion HPLC analysis of the purified YiiP-DDM-lipid micelles revealed two major chromatographic peaks (P1 and P2) with peak maxima eluted at a retention time of 16.4 and 19.2 min, respectively (Fig. 2A). Compared with the GlpF chromatograms under the identical chromatographic condition, YiiP P1 was characteristic of protein-detergent-lipid micelles with both strong UV and RI responses, whereas YiiP P2 was characteristic of empty detergent-lipid micelles with a strong RI response accompanied by a base-line level UV response. The retention time of YiiP P1 was longer than that of GlpF P1, suggesting a relatively smaller hydrodynamic radius for YiiP-detergent-lipid micelles. Molecular identities of P1 and P2 fractions were determined by MALDI-TOF mass spectrometry. P1 and P2 fractions were collected and analyzed directly in the presence of detergents and lipids. As shown in Fig. 2B, sample analyzed in this fashion yielded high quality MALDI-MS spectra over a wide range from 0.5 to 40 kDa. Because non-covalent subunit interactions were too weak to survive the MALDI process, the native YiiP oligomer was detected in the form of a monomer. Analysis of P1 fraction revealed two m/z peaks (16289.7, 32556.4) in a higher m/z range, corresponding to the expected m/z values of YiiP in double (16287.5)- and single (32575.0)-charge state. The spectrum at a lower m/z range showed two major mass species of 510.3 and 714.9, consistent with m/z values of the singly charged DDM (510.6) and an E. coli lipid that typically ranges from 700 to 800. Thus, MALDI-TOF analysis confirmed the molecular identity of P1 as mixed micelles of YiiP, DDM, and bound lipids. Analysis of the P2 fraction exhibited an m/z profile in the lower m/z range similar to that of the P1 fraction, but the expected YiiP mass peaks in the higher m/z range were absent. This result confirmed that P2 was composed of empty detergent-lipid micelles. Size-exclusion HPLC in conjunction with simultaneous UV, LS, and RI detections was used to determine molecular masses of the YiiP homo-oligomer in the mixed micelles using the instrument response factor k1 over 15 sampling points across the two chromatographic peaks, P1 (from 16.26 to 16.51 min) and P2 (from 19.05 to 19.30 min) (Fig. 2A). The mass distribution of the YiiP oligomer across P1 appeared to be homogeneous with a range from 64.6 to 70.1 kDa with an average molecular mass of 68.4 ± 1.4 kDa, suggesting that
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