Structural features and assembly of the soluble overexpressed PsaD subunitof photosystem I

PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigoclad us laminosus in Escherichia coli and obtained a soluble protein with a poly histidine tag at the carboxyl terminus. The soluble PsaD protein was purifi ed by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF. T he N-terminal amino acid sequence of the overexpressed PsaD matched the N-t erminal sequence of the native PsaD from M. laminosus. The soluble PsaD cou ld assemble into the PsaD-less PS I. As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7 x 10(6) M-1, and the enthalpy change of -93.6 kJ mol(-1). This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein. To iden tify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin- maleimide, and the biotinylated residues were mapped. The Cys(66), Lys(21), Arg(118) and Arg(119) residues were exposed on the surface of soluble PsaD whereas the Lys(129) and Lys(131) residues were not exposed on the surface . Consistent with the X-ray crystallographic studies on PS I, circular dich roism spectroscopy revealed that PsaD contains a small proportion of alpha- helical conformation. (C) 1999 Elsevier Science B.V. All rights reserved.