Abstract
Proton-translocating ATPases are central to biological energy conversion. Although eukaryotes contain specialized F-ATPases for ATP synthesis and V-ATPases for proton pumping, eubacteria and archaea typically contain only one enzyme for both tasks. Although many eubacteria contain ATPases of the F-type, some eubacteria and all known archaea contain ATPases of the A-type. A-ATPases are closely related to V-ATPases but simpler in design. Although the nucleotide-binding and transmembrane rotor subunits share sequence homology between A-, V-, and F-ATPases, the peripheral stalk is strikingly different in sequence, composition, and stoichiometry. We have analyzed the peripheral stalk of Thermus thermophilus A-ATPase by using phage display-derived single-domain antibody fragments in combination with electron microscopy and tandem mass spectrometry. Our data provide the first direct evidence for the existence of two peripheral stalks in the A-ATPase, each one composed of heterodimers of subunits E and G arranged symmetrically around the soluble A(1) domain. To our knowledge, this is the first description of phage display-derived antibody selection against a multi-subunit membrane protein used for purification and single particle analysis by electron microscopy. It is also the first instance of the derivation of subunit stoichiometry by tandem mass spectrometry to an intact membrane protein complex. Both approaches could be applicable to the structural analysis of other membrane protein complexes.
Highlights
Both subunits accumulated at high soluble levels in E. coli, and specific complexes could be isolated by copurification via Ni-nitrilotriacetic acid (NTA) affinity chromatography followed by gel filtration (Fig. 1)
Phage Display-derived Antibodies and Their Binding to the ferent domain antibody fragments (dAbs)-ATPase complexes were examined by electron microscopy (EM) in two EG Complex—Monoclonal antibodies specific to natively different approaches
In all cases the raw electron micrographs folded proteins are becoming increasingly important for struc- suggested the presence of two peripheral stalks per A-ATPase tural studies of membrane proteins because they can stabilize molecule, each containing one EG heterodimer. dAb E4, which conformations, help purification, allow labeling in EM studies, binds to the native EG heterodimer, is located at the top of the and extend the hydrophilic surface of the protein that is likely to complex, whereas D6, which recognizes preferentially subunit form ordered crystal contacts
Summary
Materials—pETDuet-1, pET-12a, and pET-25b vectors were purchased from Novagen. Taq DNA polymerase was purchased from Promega. Monoclonal phages displaying selected dAbs diluted in 2% Marvel milk powder in ATPase buffer were added into the MaxiSorp wells and incubated for 2 h. As with the phage ELISA, all subsequent incubations and washes were performed in ATPase buffer at room temperature. Antigen bound to the immobilized dAbs was detected with streptavidinalkaline phosphatase or streptavidin-horseradish peroxidase conjugate followed by development with p-nitrophenyl phosphate or tetramethylbenzidine substrate, respectively. Nondenaturing Western blotting was performed as described by Speed et al [25] using EG-specific dAbs. For denaturing SDS-PAGE analysis and Western blots, the EG complexes were mixed with NuPAGE loading buffer, boiled for 5 min, separated on 12% NuPage Bis-Tris gels, and electrotransferred onto polyvinylidene difluoride membranes. The protein concentration was ϳ40 M in 100 mM ammonium acetate, pH 7.0
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