Use of electrospray ionization mass spectrometry and tandem mass spectrometry to study binding of F0 inhibitors to ceroid lipofuscinosis protein, a model system for subunit c of mitochondrial ATP synthase.
Ceroid lipofuscinosis protein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence that is identical to that of the F0 subunit c of normal bovine ATP synthase. Electrospray ionization mass spectrometry (ESI-MS) has shown that ovine CLP and normal bovine F0 subunit c are identical, including a 42 mass unit post-translational modification. Although the identity and the location of this modification have not been fully established in both species, CLP can be used as a convenient and a unique source of subunit c for studies of F0 inhibitor interactions by ESI-MS analysis. Analysis of mixtures of CLP incubated with several known F0 inhibitors showed that N, N'-dicyclohexylcarbodiimide and organotins bind covalently to CLP but interactions with oligomycin and venturicidin were not observed. The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,-benzoquinone (UQ0) and N-ethyl maleimide (NEM) were also shown to bind covalently to the protein. The binding stoichiometry and the relative rate of reaction were then determined for each inhibitor. Tandem mass spectrometry experiments performed on the [M+5H]5+ ion of the intact CLP and of the complexes UQ0-CLP and NEM-CLP allowed the identification of 80% of the CLP sequence and revealed that UQ0 and NEM are both bound to cysteine-64. This work shows the exceptional utility of ESI-MS in studies of the interaction of CLP with a range of inhibitors which are applicable to studies of the F0 component of ATP synthase.
Buzy
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Department of Chemistry, University of Warwick, Coventry, UK.
Ryan
E M
EM
Jennings
K R
KR
Palmer
D N
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Griffiths
D E
DE
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D008247:Lysosomes / Q000201:enzymology; D013058:Mass Spectrometry; D008928:Mitochondria / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D012756:Sheep; D012997:Solvents; D013439:Sulfhydryl Reagents / Q000737:chemistry
D012997:Solvents; D013439:Sulfhydryl Reagents; D000251:Adenosine Triphosphatases; C103607:mitochondrial ATPase subunit c; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases