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The Dimerization Domain of the b Subunit of theEscherichia coli F1F0-ATPase

1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês

10.1074/jbc.274.43.31094

1083-351X

Matthew Revington, Derek T. McLachlin, Gary S. Shaw, Stanley D. Dunn,

Advanced Electron Microscopy Techniques and Applications

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F1F0 ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53–122 sequence. Sedimentation velocity and 15N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103–110 failed to form disulfides either with the identical mutant or when mixed with the other 103–110 cysteine mutants. These studies establish that the bsubunit dimer depends on interactions that occur between residues in the 53–122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface. In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F1F0 ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53–122 sequence. Sedimentation velocity and 15N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103–110 failed to form disulfides either with the identical mutant or when mixed with the other 103–110 cysteine mutants. These studies establish that the bsubunit dimer depends on interactions that occur between residues in the 53–122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface. polymerase chain reaction polyacrylamide gel electrophoresis bis(sulfosuccinimidyl)suberate nuclear Overhauser effect The F1F0 ATP synthase is a multisubunit enzyme complex that is responsible for the production of the bulk of intracellular ATP. This complex synthesizes ATP from ADP and inorganic phosphate by utilizing a transmembrane proton gradient as an energy source. Structurally and functionally the complex can be divided into two major domains: the membrane intrinsic F0 domain and the peripheral F1 domain. The F0 domain of the prototypical Escherichia coli enzyme consists of three polypeptides in the stoichiometry ofa 1 b 2 c 9–12that function in proton translocation across the inner membrane. The F1 domain, which has the subunit composition of α3β3γ1δ1ε1, performs the ATP catalytic functions. (For recent reviews of ATP synthase function and structure, see Refs. 1Boyer P. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1576) Google Scholar, 2Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar, 3Fillingame R.H. Girvin M.E. Jiang W. Valiyaveetil F. Hermolin J. Acta Physiol. Scand. Suppl. 1998; 643: 163-168PubMed Google Scholar.) Structural studies have shown that the 3α and 3β polypeptides form a ring of alternating subunits surrounding a central region occupied by the N- and C-terminal helices of the γ subunit (4Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2742) Google Scholar). The remainder of γ lies outside of the α3β3 domain and is closely associated with the ε subunit (5Tang C. Capaldi R.A. J. Biol. Chem. 1996; 271: 3018-3024Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This γε subcomplex extends for 45 Å from F1 to the membrane where interactions with the c subunits of the F0domain occur (6Watts S.D. Capaldi R.A. J. Biol. Chem. 1997; 272: 15065-15068Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Thus, γε forms the central “stalk” often seen in electron micrographs (7Lücken U. Gogol E.P. Capaldi R.A. Biochemistry. 1990; 29: 5339-5343Crossref PubMed Scopus (63) Google Scholar). The binding change model (reviewed in Ref. 1Boyer P. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1576) Google Scholar) suggests that rotation of the γε stalk relative to the α3β3domain results in sequential changes in the catalytic sites on the β subunits, causing them to cycle through conformations favoring substrate binding, then catalysis, and finally product release. This mechanism requires a stator structure to hold the α3β3 hexamer as the γε stalk rotates within it. Recently, a structure that reaches from the membrane domain to the periphery of the catalytic domain and that is distinct from the central γε stalk has been observed in electron micrographs (8Wilkens S. Capaldi R.A. Nature. 1998; 393: 29Crossref PubMed Scopus (135) Google Scholar). This “second stalk” may function as a stator formed by theb and δ subunits. The b subunit is necessary for the stable binding of F1 to the membrane and for correct assembly of the complex (9Perlin D.S. Cox D.N. Senior A.E. J. Biol. Chem. 1983; 258: 9793-9800Abstract Full Text PDF PubMed Google Scholar). b is a 156-residue polypeptide with a hydrophobic N-terminal membrane-spanning α helix. The remainder of the protein is highly polar and extends into the cytoplasm where it interacts with the δ subunit and the α3β3 domain. The cytoplasmic domain of b, consisting of residues 24–156, has been expressed separately from the membrane spanning domain and forms a soluble, highly extended homodimer that can bind F1 in a manner similar to the intact protein (10Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar). Previous studies have shown that the two b monomers in the complex can be covalently cross-linked without abolishing activity, confirming that the dimeric state is the functional form of the protein (11Rodgers A.J.W. Capaldi R.A. J. Biol. Chem. 1998; 273: 29406-29410Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The affinity ofb for F1 or the isolated δ subunit is strongly correlated with its ability to form dimers (12Sorgen P.L. Bubb M.R. McCormick K.A. Edison A.S. Cain B.D. Biochemistry. 1998; 37: 923-932Crossref PubMed Scopus (47) Google Scholar). The only significant stretch of hydrophobic amino acids in the cytoplasmic domain occurs between Val124 and Ala132. Mutation of Ala128 to aspartate has been shown to disrupt dimerization of the cytoplasmic domain (13Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Cross-linking has demonstrated that the b-δ interaction is between the C-terminal domains of each of the polypeptides (14McLachlin D.T. Bestard J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). δ can also be cross-linked to the N-terminal region of the α subunit located near the top of the of the catalytic hexamer (15Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Therefore, the second stalk is believed to be formed by the b subunits extending upward from the membrane to interact with δ, which binds on the top third of the αβ domain. The second stalk could function as a relatively passive, rigid stator or play a more active role in catalysis by transiently storing energy in an elastic manner. Much of the cytoplasmic region of the b dimer is expected to exist as a pair of extended helical rods to span the 140–150 Å from the membrane to the top of F1. In this study, we have characterized a series of N- and C-terminal truncations of the soluble b domain to identify the minimal region necessary for formation of the homodimer. In addition we have made inferences about the structure of the b dimer based on sites of intersubunit disulfide formation and on changes in hydrodynamic behavior upon truncation of the sequence. Molecular biological procedures were carried out as described by Sambrook et al. (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Constructions were confirmed by restriction endonuclease mapping and regions of DNA derived from PCR1 products were sequenced to ensure that only the desired mutations had been introduced. Plasmid pDM3, which carries a synthetic sequence encodingb 24–156 2b x–y indicates a polypeptide containing the amino acid sequence of b from positionx to position y, generally preceded by a short leader sequence derived from the plasmid. For example,b 24–156 refers to a construct consisting of residues of Tyr24 to Leu156 of the bsubunit. Assuming removal of the N-terminal methionine, those constructs beginning with Tyr24 have a leader sequence of TMITNSH, whereas all other constructs used in this study have a leader sequence of SYW (Fig. 1).in pUC8, has been described previously (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To stabilize mRNAs and enhance expression of various forms of b, a 202-base pairBfaI-HindIII fragment of pSD100 (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) carrying theunc transcriptional terminator was inserted downstream of the b 24–156 synthetic gene, using theHindIII and NdeI sites. The resulting plasmid was called pDM3T. A number of plasmids encoding N- or C-terminal truncations of the soluble form of b were used in this work; these plasmids and polypeptides are summarized in Fig. 1. Previously, work from this laboratory described three plasmids, pSD114, pSD111, and pKK1, which express soluble b polypeptides lacking 33, 52, or 66 residues, respectively, from the N-terminal of b (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). For the current studies a series of plasmids encoding C-terminal truncations was constructed by PCR mutagenesis. In these constructions, PCR using plasmid pDM3 as a template was carried out using the M13 reverse universal primer coupled with mutagenic primers containing aHindIII restriction site and a sequence complementary to a stop codon and the desired region of synthetic b sequence. PCR products were co-digested with HindIII and a second enzyme, both unique in plasmids pDM3 or pDM3T, and inserted into one of those plasmids using the same restriction sites. Sequences encoding some of the C-terminal truncations were pasted into pSD114 or pSD111 to produce forms of soluble b bearing deletions at both ends. Another set of plasmids was constructed; each of these plasmids encoded a mutant form of b 24–156 in which one of the amino acid residues between Ala103 and Glu110of the normal b sequence was replaced by cysteine. The residue numbers cited refer to the position in the wild typeb sequence. These plasmids were constructed by PCR mutagenesis and cloned into pDM3 using standard techniques as described previously (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Induced cells expressing the polypeptide of interest were suspended in a volume of 50 mmTris-HCl, pH 8.0, 10 mm MgCl2, 1 mmphenylmethylsulfonyl fluoride equal to 10 times their packed wet weight and disrupted by one passage through a French pressure cell at 20,000 p.s.i. Cell debris was removed by centrifugation for 10 min at 9,000 rpm in a Beckman JA-20 rotor. The supernatant solution was centrifuged for another 1.5 h at 38,000 rpm in a Beckman Ti-60 rotor. The purification of polypeptides b 24–156,b 34–156, b 53–156,b 24–152, and b 67–156have been described previously (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Other b truncated polypeptides were purified from the high speed supernatant solutions using generally similar techniques of ammonium sulfate precipitation, ion exchange chromatography, and size exclusion chromatography, with the modifications summarized below. During purification, fractions were analyzed by SDS-PAGE, and the final products were essentially pure, as judged by this technique. b 24–145 was precipitated with 40% saturated ammonium sulfate, redissolved and dialyzed against TE buffer (50 mm Tris-HCl, pH 8.0, 1 mm EDTA). The protein was loaded onto a DEAE-Sepharose column equilibrated with TE buffer and eluted with a linear gradient of 0–250 mm NaCl in TE buffer. Fractions containing the protein were again dialyzed and loaded onto a second DEAE-Sepharose column; this second column was eluted with a gradient of 0–200 mm NaCl in 25 mmimidazole-HCl buffer at pH 6.4. The protein was precipitated with 70% saturated ammonium sulfate and further purified by size exclusion chromatography on a column of Sephacryl S-300. b 24–134 and b 24–138were both purified using the following procedure. The proteins were precipitated with 40% saturated ammonium sulfate, redissolved, and dialyzed against TE buffer. Upon application to a column of DEAE-Sepharose equilibrated with TE buffer, the proteins did not bind but upon elution with TE buffer were retarded slightly. This step was repeated on a second column of DEAE-Sepharose to remove yet more impurities. The protein was precipitated with ammonium sulfate and finally purified by size exclusion chromatography on a column of Sephacryl S-300. b 24–122 was precipitated with 35–55% saturated ammonium sulfate, dialyzed against TE buffer, and run through two columns of DEAE-Sepharose, similar to the procedure described forb 24–134 and b 24–138, to remove other proteins. After precipitation with ammonium sulfate, final purification was by size exclusion chromatography on a column of Sephadex G-75. b 24–114 was unlike the other polypeptides described above in that a part of the protein was insoluble and precipitated during low speed centrifugation. The purification was continued with the portion of the protein that remained in the supernatant. Following high speed centrifugation, theb 24–114 was precipitated from the supernatant by adding ammonium sulfate to a concentration of 55% saturation. The precipitate was redissolved in TE buffer and dialyzed, and most contaminating proteins were removed by passage through a column of DEAE-Sepharose, as for b 24–134 andb 24–138. Appropriate fractions were pooled, the protein was precipitated with ammonium sulfate, and final purification was by size exclusion chromatography on a column of Sephacryl S-200. b 34–122 was precipitated with 45% saturated ammonium sulfate, dialyzed against TE buffer, and run through a column of DEAE-Sepharose, similar to the procedure described forb 24–134 and b 24–138. Appropriate fractions were pooled and adjusted to pH 4.8 by addition of acetic acid. Final purification was achieved by loading the sample on a column of CM-Sepharose and elution with a linear gradient of 0–1m NaCl in 25 mm sodium acetate buffer, pH 4.8. b 53–122 was precipitated with 45–70% saturated ammonium sulfate and run through two columns of DEAE-Sepharose, similar to the procedure described forb 24–134 and b 24–138. Appropriate fractions from the second column were pooled, and the pH was adjusted to 5.0 by the addition of acetic acid. Final purification was achieved by loading the sample onto a column of CM-Sepharose equilibrated with 30 mm sodium acetate buffer, pH 5.0, and elution with a linear gradient of 100–600 mm NaCl in 30 mm sodium acetate, pH 5.0. Protein purity was assessed by 15% SDS-PAGE gels using standard glycine running buffers except for the constructs of molecular weight less than 10 kDa (b 34–122,b 53–122) where improved resolution was obtained using 15% Laemmli gels (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206602) Google Scholar) but with a running buffer of 100 mm Tris, 100 mm Tricine, 0.1% SDS. All chemicals and solvents used were reagent grade. Protein concentrations were determined spectrophotometrically at 280 nm exceptb 67–156, which lacked an aromatic chromophore and was observed at 240 nm. The extinction coefficients at 280 and 240 nm were based on concentrations determined by quantitative amino acid analysis. Purified protein samples were dialyzed into 50 mm Tris-HCl, pH 7.5, 0.1m NaCl, 1 mm EDTA, or 50 mm sodium acetate, pH 5.0 for ultracentrifugal analyses. Initial concentrations of the polypeptides analyzed were between 20 and 50 μm of monomer. Unless otherwise noted all reported concentrations refer to monomer. Sedimentation equilibrium data were collected in a Beckman Optima XL-A Analytical Ultracentrifuge with absorbance optics. A four-hole An-60Ti rotor and six-channel cells with Epon charcoal centerpieces were used. Absorbance measurements were taken in 0.002-cm radial steps and averaged over 10 observations. Samples were allowed to equilibrate for 24 h at the desired speed (30,000 or 36,000 rpm) and temperature (5, 20, or 40 °C) before scanning. After the 40 °C scan the samples were cooled to 20 °C, allowed to equilibrate, and scanned again to ensure the reversibility of the monomer-dimer transition and to confirm that no degradation had occurred. Software supplied by Beckman was used for data processing and curve fitting. The reported observed molecular weight,M obs, for a single species was calculated based on the average of three data sets. The monomer molecular weight,M c, was calculated from the amino acid sequence. The partial specific volumes of proteins were determined from the amino acid sequence by the method of Cohn and Edsall (19Cohn E.J. Edsall J.T. Proteins, Amino Acids, and Peptides. Reinhold, New York1943: 157-161Google Scholar). The density of the solvents were measured in a pycnometer or, in some cases, calculated from published tables (20Weast R.C. CRC Handbook of Chemistry and Physics. Chemical Rubber Company, Cleveland, OH1970: D-176-D-215Google Scholar). Sedimentation velocity samples were run at 60,000 rpm in double-sector cells at 5 and 20 °C. Once full speed was attained the samples were scanned every 10 min for a total run time of up to 300 min. Each scanned point was the average of five observations, and all data reported were the averages of at least three runs. The data sets were processed using the SVEDBERG program (21Philo J.S. Biophys. J. 1996; 72: 435-444Abstract Full Text PDF Scopus (212) Google Scholar) utilizing the modified Fujita-MacCosham model fitted for a single species. When necessary the observed values for sedimentation coefficients,s obs, were converted tos 20,w. Frictional coefficients (f) and frictional ratios (ƒ/ƒmin) were calculated fromM r and s 20,w by standard methods. NMR spectra were collected on a Varian Unity 500 MHz spectrometer equipped with a triple resonance probe and z axis pulsed field gradients. A sample of15N-labeled b 53–122 in 50 mm acetate buffer, pH 5.0, was equilibrated at 25 °C. One-dimensional T1, T2, and 15N NOE spectra were collected using the pulse sequences described in Farrow et al. (22Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.F. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2002) Google Scholar). The T1 relaxation was measured in an array of 25 steps from 11.2 ms to 1.36 s in 56-ms increments. The T2 signal was measured from an array of 11 spectra with relaxation delays from 16.6 to 183.6 ms in 16.6-ms steps. For the T1 and T2 data the signal envelopes were integrated and fit to determine the first order exponential decay constant. The NOE experiments were run with either a 5-s relaxation delay for the no NOE base-line experiment or a relaxation delay of 2 s followed by a 3-s 1H presaturation period to determine the magnitude of 15N NOE. The ratios between the intensities of selected peaks in the NOE and no NOE experiments were calculated. Global correlation times, τm, were calculated as described previously by Farrow et al. (22Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.F. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2002) Google Scholar). The cross-linking ofb 53–122, b 67–156,b 24–114, and b 34–156was carried out for 10 min at room temperature using 1 mmbis(sulfosuccinimidyl)suberate (BS3) (Pierce). The cross-linking reactions occurred in the presence of 50 mmtriethanolamine buffer, 1 mm EDTA, pH 7.5, and were quenched by the addition of ethanolamine-HCl, pH 7.5, to a final concentration of 100 mm. Complete quenching was achieved by leaving the reactions standing for 10 min at room temperature followed by heating in SDS sample buffer. The products of the reactions were then run on 15% SDS-PAGE gels using the Tricine running buffers previously specified. Forms ofb 24–156 bearing mutations incorporating cysteine into one of the positions between Ala103 and Glu110 were partially purified and tested for their ability to form disulfide bonds as described previously (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, the partially purified proteins were reduced by dialysis into buffer containing 1 mm dithiothreitol and then dialyzed in buffer containing 0.1 m NaHCO3, 10 μmCuCl2, and 10 mm cysteine at 4 °C to induce disulfide bond formation. After 24 h, samples were treated with 15 mm N-ethyl maleimide in SDS-PAGE sample buffer to block unreacted thiol groups and analyzed by nonreducing SDS-PAGE. Previous and current studies of the b subunit suggest a four-domain model, which is shown in Fig.1 A to assist in the presentation of data. The N-terminal 24-residue sequence is highly hydrophobic and is embedded in the membrane with the other F0 subunits. The sequence from residues 25 to 52, which is not essential for dimer formation (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), is designated the tether domain, because it joins the membrane region to the beginning of the sequence essential for dimerization. The extent of the remaining sequence that is necessary for homodimer formation is defined in this report. Near the C terminus are sites required for binding to the δ subunit and the F1 sector; we refer to this region as the δ-binding domain. Previous work from this laboratory has characterized soluble forms of b lacking the N-terminal membrane spanning domain (10Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar), three deeper N-terminal deletions (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and minor C-terminal truncations (14McLachlin D.T. Bestard J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In the current work, a series of plasmids encoding deeper C-terminal truncations of b was constructed (Fig. 1), and the polypeptides were purified and characterized. All of the new forms were extracted as soluble polypeptides, exceptb 24–114, which was partly soluble and partly inclusion body. The purification of these proteins by conventional techniques of ammonium sulfate precipitation, ion exchange chromatography, and size exclusion chromatography is described under “Experimental Procedures.” A number of properties of these polypeptides became apparent during this work. All C-terminal deletions redissolved easily following ammonium sulfate precipitation, unlike forms with an intact C terminus. Little difference in the percentage of ammonium sulfate required for precipitation was apparent, however, until the hydrophobic region between residues 124 and 132 was removed. Forms truncated at residue 138 or earlier in the sequence failed to bind to DEAE resin at pH 8.0, underscoring the acidic nature of the C-terminal region that had been removed. There are six acidic and two basic side chains between 134 and 156; deletion of this region changes the pI for the protein from about 6 to over 8. DEAE-Sepharose was nevertheless used during the purification of these polypeptides, because it absorbed most of the other proteins in the samples. The less acidic nature of these proteins resulted in a much enhanced solubility at pH 5.0 in comparison with forms like b 34–156, which precipitate as the pH is lowered toward 5.0 (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This property was exploited through purification on CM-Sepharose. Sedimentation equilibrium centrifugation allows accurate determination of the molecular weight of soluble proteins under native conditions and hence the stoichiometry of multimeric complexes. Previous studies of the b 24–156construct have demonstrated that the isolated cytoplasmic domain exists primarily as a dimer (10Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar). In this study of the molecular weight of the b subunit truncations, sedimentation equilibrium data were fitted to a single component model to determine the average observed molecular weight,M obs, of the purified protein in solution. The data are presented in Table I asM obs/M c, the ratio ofM obs to the molecular weight of the monomer calculated from the sequence, M c. The presence of equilibrium mixtures of monomers and dimers is reflected in the nonintegral values for these ratios.Table IOligomerization of the b polypeptides determined by sedimentation equilibrium analysisPolypeptideMcMobsaMc5 °C20 °C40 °CCytoplasmic domain of b b24–15615,5081.87 ± 0.062.11 ± 0.081.39 ± 0.06N-terminal truncations b34–15614,0181.88 ± 0.062.13 ± 0.111.36 ± 0.04 b53–15611,8982.02 ± 0.072.40 ± 0.021.71 ± 0.12 b67–1569,8631.08 ± 0.041.18 ± 0.031.10 ± 0.05C-terminal truncations b24–15214,9331.81 ± 0.011.89 ± 0.011.16 ± 0.03 b24–13813,6391.81 ± 0.051.81 ± 0.021.29 ± 0.03 b24–12211,9761.79 ± 0.041.76 ± 0.031.09 ± 0.01 b24–11410,7741.29 ± 0.081.16 ± 0.011.08 ± 0.01N- and C-terminal truncations b53–1228,3651.96 ± 0.051.89 ± 0.031.25 ± 0.03 b53–122, pH 5.08,3651.95 ± 0.021.99 ± 0.021.39 ± 0.01The sedimentation equilibrium experiments were carried out at pH 7.5 in 50 mm Tris-HCl, 100 mm NaCl, 1 mmEDTA buffer except where pH 5.0 is noted. In those studies a 50 mm sodium acetate buffer was employed.a The M obs/M cvalues reported are the average and standard error of three experiments. Open table in a new tab The sedimentation equilibrium experiments were carried out at pH 7.5 in 50 mm Tris-HCl, 100 mm NaCl, 1 mmEDTA buffer except where pH 5.0 is noted. In those studies a 50 mm sodium acetate buffer was employed. a The M obs/M cvalues reported are the average and standard error of three experiments. In previous work it was demonstrated thatb 24–156, b 34–156, andb 53–156 exist primarily as dimers, whereas theb 67–156 truncation was monomeric in solution (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In the current work an analysis of the effect of temperature on the properties of these polypeptides was undertaken. The average oligomerization states of the solution species at 5, 20, and 40 °C are listed in Table I. Comparison of the values at 5 and 20 °C for the three dimeric constructs revealed that there was a trend for theM obs/M c ratio to increase from about 1.9 at 5 °C to slightly more than 2 at 20 °C. We have previously reported that b 24–156 has a slight tendency to aggregate at 20 °C when its concentration is higher than about 1 mg/ml (17McLachlin D.T. Dunn S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Because the values reported in Table I reflect average molecular weights of species present in the ultracentrifuge cell, this slight aggregation can raise theM obs/M c ratio to values above 2.0. At 40 °C, theM obs/M c ratio dropped to less than 1.4 for b 24–156 andb 34–156, indicating that the higher temperatures destabilized the dimer interaction, shifting the monomer-dimer equilibrium toward the monomeric state. Theb 53–156 polypeptide gave higher values than the less truncated forms b 24–156 andb 34–156 at all temperatures examined; theM obs/M c value of 2.40 obtained at 20 °C for this construct indicates that it is especially prone to aggregate to species higher than dimer. It is likely that the value obtained at 40 °C, 1.71, was higher than those forb 24–156 and b 34–156because of the effect of this aggregation on the average molecular weight, rather than because removing residues 34–52 caused the remainder of the protein to form a tighter dimer. As expected, theb 67–156 construct exhibitedM obs/M c values below 1.2 for the entire tempe