Ca2+ binding effects on the C2 domain conformation of human cytosolic phospholipase A2.

It has been reported that the cooperative binding of calcium ions indicated a local conformational change of the human cytosolic phospholipase A2 (cPLA2) C2 domain (Nalefski et al., (1997) Biochemistry 36, 12011-12018). However its structural evidence is less known (Malmberg et al., (2003) Biochemistry 42, 13227-13240). In this letter, life-time decay and fluorescence quenching techniques were employed to compare the calcium-induced conformational changes. The life-time decay parameters and fluorescence quenching constant changes were small between the apo- and holo-C2 domains when tryptophan residue was excited at 295 nm. In contrast, the quenching constant change was large, from 0.52 M(-1) for the apo-C2 to 8.8 M(-1) for the holo-C2 domain, when tyrosine residues were excited at 284 nm. Our results provide new information on amino acid side chain orientation change at calcium binding loop 3, which is necessary for Ca2+ binding regulated membrane targeting of human cytosolic phospholipase A2.

Sequential conformation changes of chinese hamster ovary dihydrofolate reductase at its active sites.

The local fluorescence probes, 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and NADPH were employed to detect urea-induced conformation changes at each active site of dihydrofolate reductase (DHFR), respectively. The results indicate that local conformation change at DHF/TNS could be superimposed by the conformation change calculated from the enzyme activity change with a three-state model; while at NADPH site it is lagged in the first transition. This difference is further supported by the different relative changes of Michaelis constants at 0, 1 and 1.8 M urea for each substrate. Our results suggest that local conformation at DHF site is more flexible than that at NADPH site, and the urea-induced unfolding could be ascribed to a four-state transition.