Characterisation of subunit III and its oligomer from spinach chloroplast ATP synthase

• Published in: Biochimica et biophysica acta 2003-12, Vol.1618 (1), p.59-66
• Main Authors: Poetsch, Ansgar, Rexroth, Sascha, Heberle, Joachim, Link, Thomas A., Dencher, Norbert A., Seelert, Holger
• Format: Article
• Language: English
• Subjects: CD spectroscopy; Chloroplast Proton-Translocating ATPases - chemistry; Chloroplast Proton-Translocating ATPases - isolation & purification; Chloroplast Proton-Translocating ATPases - metabolism; Circular Dichroism; FTIR; MALDI mass spectrometry; Membrane protein; Protein Structure, Secondary; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spinacia oleracea - enzymology
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2003.10.007

Proton ATP synthases carry out energy conversion in mitochondria, chloroplasts, and bacteria. A key element of the membrane integral motor CF O in chloroplasts is the oligomer of subunit III: it converts the energy of a transmembrane electrochemical proton gradient into rotational movement. To enlighten prominent features of the structure–function relationship of subunit III from spinach chloroplasts, new isolation methods were established to obtain highly pure monomeric and oligomeric subunit III in milligram quantities. By Fourier-transform infrared (FTIR) and CD spectroscopy, the predominantly α-helical secondary structure of subunit III was demonstrated. For monomeric subunit III, a conformational change was observed when diluting the SDS-solubilized protein. Under the same conditions the conformation of the oligomer III did not change. A mass of 8003 Da for the monomeric subunit III was determined by MALDI mass spectrometry (MALDI-MS), showing that no posttranslational modifications occurred. By ionisation during MALDI-MS, the noncovalent homooligomer III 14 disaggregated into its III monomers.