STUDY OF THE CONFORMATION OF DARPP-32, A DOPAMINE-REGULATED AND CAMP-REGULATED PHOSPHOPROTEIN, BY FLUORESCENCE SPECTROSCOPY

DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is pho sphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is als o phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. W e have studied the conformation of recombinant rat DARPP-32 by steady- state and time-resolved fluorescence. The steady-state emission spectr a and quenching of the intrinsic (Trp163) and extrinsic fluorescence ( acrylodan or lucifer yellow linked to Cys72) were consistent with a co mplete exposure of these residues to the aqueous environment. The intr insic fluorescence of DARPP-32 was resolved into three decay component s with lifetimes of 1, 3.4, and 7 ns, with the intermediate lifetime c omponent giving the major contribution. The ratio between the amplitud es associated with the short and long decay constants was decreased up on denaturation. The rotational behavior of DARPP-32 measured by aniso tropy decay revealed that Trp163 is located in a highly flexible pepti de chain, whereas Cys72 is embedded in a more rigid environment. Phosp horylation by cAMP-dependent protein kinase did not alter any of the f luorescence parameters, whereas only minor effects were associated wit h casein kinase II phosphorylation. These findings indicate that DARPP -32 contains at least two distinct domains and that phosphorylation ha s no dramatic effects on its conformation.