Cross-linking of engineered subunit delta to (alphabeta)3 in chloroplast F-ATPase.
Ser --> Cys mutations were introduced into subunit delta of spinach chloroplast F0F1-ATPase (CF0CF1) by site-directed mutagenesis. The engineered delta subunits were overexpressed in Escherichia coli, purified, and reassembled with spinach chloroplast F1-ATPase (CF1) lacking the delta subunit (CF1(-delta)). By modification with eosin-5-maleimide, it was shown that residues 10, 57, 82, 160, and 166 were solvent-accessible in isolated CF1 and all but residue 166 also in membrane-bound CF0CF1. Modification of the engineered delta subunit with photolabile cross-linkers, binding of delta to CF1(-delta), and photolysis yielded the same SDS gel pattern of cross-link products in the presence or absence of ADP, phosphate, and ATP and both in soluble CF1 and in CF0CF1. By chemical hydrolysis of cross-linked CF1, it was shown that deltaS10C was cross-linked within the N-terminal 62 residues of subunit beta. deltaS57C, deltaS82C, and deltaS166C were cross-linked within the N-terminal 192 residues of subunit alpha. Cross-linking affected neither ATP hydrolysis by soluble CF1 nor its ability to reassemble with CF0 and to structurally reconstitute ATP synthesis. Functional reconstitution, however, seemed to be impaired.
Lill
H
H
Biophysik, Fachbereich Biologie/Chemie, Universität Osnabrück, Barbarastrasse 11, D-49069 Osnabrück, Federal Republic of Germany. engel@ax3301.biologie.Uni-Osnabrueck.DE
Hensel
F
F
Junge
W
W
Engelbrecht
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D046911:Macromolecular Substances; D008958:Models, Molecular; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000737:chemistry
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine