Recognition of partially-folded mitochondrial malate dehydrogenase by GroEL. Steady and time-dependent emission anisotropy measurements

The binding of partially-folded mitochondrial malate dehydrogenase (mMDH) t o GroEL was assessed by steady and nanosecond emission spectroscopy. Partia lly-folded intermediates of mMDH show significant residual secondary struct ure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein-ANS complexes display a rotational c orrelation time of 19 ns. Similar rotational correlation time (phi = 18.6 n s) was determined for partially-folded species tagged with anthraniloyl. Gr oEL recognizes partially-folded species with a K-D similar or equal to 60 n M. The rotational correlation time of the complex, i.e., GroEL-mMDH-ANT, ap proaches a value of 280 ns in the absence of ATP. Reactivation of mMDH-ANT by addition of GroEL and ATP brings about a significant decrease in the obs erved rotational correlation time. The results indicate that partially-fold ed malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conf ormations endowed with catalytic activity.