Fluorescence resonance energy transfer between coumarin-derived mitochondrial F(1)-ATPase gamma subunit and pyrenylmaleimide-labelled fragments of IF(1) and c subunit.
We introduced a reporting group into a critical position of the mitochondrial F(1)-ATPase in order to gain structural information about enzyme-ligand complexes. Incubation of 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) with bovine heart mitochondrial F(1)-ATPase pretreated with 1 nM sodium arsenite modified the only cysteine residue in the gamma subunit (gamma-Cys(78)), resulting in an enzyme-CPM fluorescent complex (CPM-F(1)) with an ATPase activity similar to that of the native enzyme. Transferred fluorescence of F(1)-bound CPM occurred when different peptide fragments of naturally binding polypeptides carrying a pyrenylmaleimide (PM) moiety were bound to the enzyme. Fluorescence resonance energy transfer (RET) from PM bound to cysteine residues associated with Glu(40), Lys(47) and Lys(58) of fragments of the inhibitor protein (IF(1)) with CPM-F(1) occurred with an efficiency of approx. 20, 21 and 3% respectively. The distance at which the efficiency of energy transfer was 50%, R(0), for the CPM and PM donor/acceptor pair was 4.1 nm, indicating that the three IF(1) fragments must be within 6.7 nm of gamma-Cys(78). RET from the PM-bound hydrophilic fragment of c subunit (residues 37-42) of the F(1)F(0)-ATPase complex and CPM-bound gamma-Cys(78) occurred with an efficiency of approx. 30%, indicating a distance of 4.7 nm between the two fluorophores. Based on previous observations and on the present RET measurements, the hydrophilic loop of c subunit was located at the base of the F(1) foot, and the N-terminal region of IF(1) was located on the surface of F(1) in the lower part of the alpha(3)beta(3) hexamer ring.
Baracca
Alessandra
A
Dipartimento di Biochimica G. Moruzzi, Università degli Studi di Bologna, via Irnerio 48, 40126 Bologna, Italy.
Barogi
Silvia
S
Paolini
Sara
S
Lenaz
Giorgio
G
Solaini
Giancarlo
G
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003374:Coumarins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry
D003374:Coumarins; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D006180:Proton-Translocating ATPases