NMR solution structure of the mitochondrial F1β presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F(1)beta subunit from Nicotiana plumbaginifolia. A general method for purification of pre-sequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F(1)beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F(1)beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (similar to50% alpha-helix), and in acidic phospholipid bicelles (similar to60% alpha-helix). The NMR solution structure of the F(1)beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site. (C) 2004 Elsevier Ltd. All rights reserved.