D T McLachlin

The University of Western Ontario, London, Ontario, Canada

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Publications (10)42.03 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensional gel electrophoresis confirmed binding of the modified b mutants to F1-ATPase as well as to F1-delta. Subsequent addition of delta to F1-delta.b2 complex resulted in changes in the ESR spectra, indicating different binding interactions of b to F1 in the presence or absence of delta. The data also suggest that the reconstitution of the ATP synthase is not ordered with respect to these subunits. Additional spectral components observed in b preparations that were spin-labeled between amino acid position 101 and 114 are indicative of either two populations of b subunits with different packing interactions or to helical bending within this region.
    Journal of Biological Chemistry 12/2004; 279(47):49074-81. · 4.65 Impact Factor
  • D T McLachlin, A M Coveny, S M Clark, S D Dunn
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    ABSTRACT: The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.
    Journal of Biological Chemistry 07/2000; 275(23):17571-7. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The b subunit dimer of theEscherichia coli ATP synthase, along with the δ subunit, is thought to act as a stator to hold the α3β3 hexamer stationary relative to thea subunit as the γεc 9–12complex rotates. Despite their essential nature, the contacts betweenb and the α, β, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound bsubunit, or within b 24–156, a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F1 sector or to complete F1F0 was attempted. Cross-linking in both the full-length and truncated forms ofb was obtained at positions 92 (to α and β), and 109 and 110 (to α only). Mass spectrometric analysis of peptide fragments derived from the b 24–156A92C cross-link revealed that cross-linking took place within the region of α between Ile-464 and Met-483. This result indicates that the b dimer interacts with the α subunit near a non-catalytic α/β interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F0 in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain ofb are adjacent to residues of a. Sites of cross-linking between b 24–156A92C and β as well as b 24–156I109C and α are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.
    Journal of Biological Chemistry 06/2000; 275(23):17571-17577. · 4.65 Impact Factor
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    S D Dunn, D T McLachlin, M Revington
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    ABSTRACT: Two stalks link the F(1) and F(0) sectors of ATP synthase. The central stalk contains the gamma and epsilon subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic alpha(3)beta(3) complex. The two b subunits and the delta subunit associate to form b(2)delta, a second, peripheral stalk extending from the membrane up the side of alpha(3)beta(3) and binding to the N-terminal regions of the alpha subunits, which are approx. 125 A from the membrane. This second stalk is essential for binding F(1) to F(0) and is believed to function as a stator during rotational catalysis. In vitro, b(2)delta is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with delta. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one alpha subunit or alphabeta pair. However, the weak interactions between the polypeptides in b(2)delta pose a challenge for the proposed stator function.
    Biochimica et Biophysica Acta 06/2000; 1458(2-3):356-63. · 4.66 Impact Factor
  • D T McLachlin, S D Dunn
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    ABSTRACT: The ATP synthase of Escherichia coli is believed to act through a rotational mechanism in which the b(2)delta subcomplex holds the alphabeta hexamer stationary relative to the rotating gamma and epsilon subunits. We have engineered a disulfide bond between cysteines introduced at position 158 of the delta subunit and at a position just beyond the normal C-terminus of the b subunit. The formation of this disulfide bond verifies that the C-terminal region of b is proximal to residue 158 of delta. The disulfide bond does not affect the ability of the F(1)F(0) complex to hydrolyze ATP, couple ATP hydrolysis to the establishment of a proton gradient, or maintain a proton gradient generated by the electron transport chain. These results are consistent with a permanent association of b(2) with delta as suggested by the rotational model of enzyme function.
    Biochemistry 04/2000; 39(12):3486-90. · 3.38 Impact Factor
  • M Revington, D T McLachlin, G S Shaw, S D Dunn
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    ABSTRACT: In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) 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 (15)N 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 b subunit 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.
    Journal of Biological Chemistry 11/1999; 274(43):31094-101. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of Biological Chemistry 10/1999; 274(43):31094-31101. · 4.65 Impact Factor
  • D T McLachlin, J A Bestard, S D Dunn
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    ABSTRACT: An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta subunit of the enzyme. To determine whether these residues interact directly with delta, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and delta were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and delta subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.
    Journal of Biological Chemistry 07/1998; 273(24):15162-8. · 4.65 Impact Factor
  • D T McLachlin, S D Dunn
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    ABSTRACT: Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactions in the b subunit of Escherichia coli F1F0-ATPase. Individual cysteine residues were incorporated into bsyn, a soluble form of the protein lacking the membrane-spanning N-terminal domain, in two main areas: the heptad repeat region and the hydrophobic region which begins at residue Val-124. The tendencies of these cysteine residues to form disulfide bonds with the corresponding cysteine in the bsyn dimer were tested using disulfide exchange by glutathione and air oxidation catalyzed by Cu2+. Within the heptad repeat region, only cysteines at residues 59 and 60, which occupy the b and c positions of the heptad repeat, showed significant tendencies to form disulfides, a result inconsistent with a coiled-coil model for bsyn. Mixed disulfide formation most readily occurred with the S60C + L65C and A61C + L65C pairs. Cysteines at positions 124, 128, 132, and 139 showed strong tendencies to form disulfides with their mates in the dimer, suggesting a parallel alpha-helical interaction between the subunits in this region. Deletion of residues N-terminal to either Glu-34 or Asp-53 had no apparent effect on dimerization as determined by sedimentation equilibrium, while deletion of all residues N-terminal to Lys-67 produced a monomeric form. These results imply that residues 53-66 but not 24-52 are essential for bsyn dimerization. Taken together the results are consistent with a model in which the two b subunits interact in more than one region, including a parallel alignment of helices containing residues 124-139.
    Journal of Biological Chemistry 09/1997; 272(34):21233-9. · 4.65 Impact Factor
  • D T McLachlin, S D Dunn
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    ABSTRACT: A method of screening transformed bacterial colonies for introduction of a cysteine residue into an overexpressed protein is described. After treating SDS extracts of induced bacterial cells with fluorescein-5-maleimide, the proteins containing cysteine were visible on SDS-PAGE gels under ultraviolet light as fluorescent bands. If the wild-type protein contains no endogenous cysteine residues, then mutant proteins containing cysteine may be easily identified by their fluorescence. In addition, a shift in electrophoretic mobility of modified proteins was observed, with mutant proteins containing cysteine at more than one site exhibiting incremental decreases in electrophoretic mobility. This effect permits the detection of cysteine mutations even when endogenous cysteines are present. The described method allows the rapid screening of a large number of transformants.
    Protein Expression and Purification 06/1996; 7(3):275-80. · 1.43 Impact Factor