Limited Proteolysis Study of the Structure of Chloroplast Coupling Factor CF₁

A. N. Malyan^*, O. I. Vitseva, O. N. Gubanova, and T. A. Muranova

Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (0967) 79-0532; E-mail: Malyan@issp.serpukhov.su

^* To whom correspondence should be addressed.

Received June 18, 1998; Revision received July 20, 1998

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The structure of chloroplast coupling factor CF₁ was studied by limited proteolysis followed by sodium dodecylsulfate polyacrylamide gel electrophoresis and N-terminal sequence analysis. The N-terminal fragment of the alpha-subunit was shown to have an exposed area including the peptide bonds E17--G18, R21--E22, E22--V23, and K24--V25. The cleavage of peptide bonds at amino acid residues E22 or/and V25 caused weaker subunit interactions and partial dissociation of the alpha- and gamma-subunits. In the N-terminal fragment of the isolated CF₁ beta-subunit, the L14--E15 bond was found to be exposed to proteolytic attack. Also, the alphaS86--S87, alphaE125--S126, alphaR127--L128, and betaV76--A77 bonds were subject to proteolysis. The correlation between the accessibility of these bonds to proteases and the surface location of similar bonds in mitochondrial F₁ was deduced from its molecular structure and a computed model of CF₁. The K204--C205 bond exposed to protease on reduction of the CF₁ gamma-subunit was shown to be masked when the catalytic reaction was initiated by CaATP.

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KEY WORDS: chloroplasts, coupling factor CF₁, ATPase, ATP synthase, protein structure, proteolysis

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