Conformational analysis of a mitochondrial presequence derived from the F 1-ATPase β-subunit by CD and NMR spectroscopy

Abstract

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the prequence for the β-subunit of F 1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, α-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and α-helical conformations, with a significant population of α-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4–10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the α-helical content to increase even further, and the TFE titration data for the presequence peptide of the F 1-ATPase β-subunit are not consistent with a single, cooperative transition from random coil to α-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F 1-ATPase β-subunit.