NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia.
We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences 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 F1beta 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 F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta 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.
Moberg
Per
P
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Nilsson
Stefan
S
Ståhl
Annelie
A
Eriksson
Anna-Carin
AC
Glaser
Elzbieta
E
Mäler
Lena
L
D004792:Enzyme Precursors / Q000302:isolation & purification; D008823:Micelles; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000737:chemistry; D012996:Solutions; D014026:Nicotiana / Q000201:enzymology*
D004792:Enzyme Precursors; D008823:Micelles; D021122:Protein Subunits; D012996:Solutions; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases