Nucleotide Binding to Chloroplast ATP Synthase: Effect on the Proton Spin-Echo NMR Spectrum

R. R. Sharp, W. D. Frasch

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Effects of nucleotide binding on the high-resolution proton spin-echo spectrum of chloroplast ATP synthase [coupling factor 1 (CF1] have been studied. Spin-echo difference spectra obtained at an 18-ms pulse spacing have been recorded ± stoichiometric amounts of 5'-adenylyl β-imidodiphpsphate (AMPPNP), a nonhydrolyzable substrate analogue. Addition of Mg-AMPPNP to solutions of causes a highly specific shortening of T2 of two proton resonances at 2.70 and 2.29 ppm, which have previously been assigned to β- and γ-methylene protons of aspartate and glutamate, respectively, on the basis of studies of the effects of covalent modification of carboxyl-bearing side chains on CF1. The observed T2 shortening, which indicates decreased mobility of these side chains in the presence of nucleotide, results from nucleotide binding to a tight side (Kd ≈ 10-6 M) which is present at a mole ratio of 1 mol of nucleotide per mole of CF]. Parallel experiments have also been conducted with the mangano-AMPPNP complex, which is paramagnetic and can produce additional relaxation enhancements of neighboring protons by means of the through-space nuclear dipole-electron dipole interaction. The effective range of this interaction in the present experiments is estimated to be at least 19 Å but no greater than 25 Å. From a comparison of relaxation enhancements produced by Mg-AMPPNP with those produced by Mn-AMPPNP, it is concluded that the majority of the resonances visible in the 18-ms spin-echo NMR spectrum lie outside the dipolar sphere of influence. The aspartate and glutamate resonances which are immobilized by Mg-AMPPNP binding do not coordinate directly to the metal ion but lie near the periphery of this sphere.

Original languageEnglish (US)
Pages (from-to)5454-5458
Number of pages5
Issue number20
StatePublished - Sep 1 1985
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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