title: Recognition of partially-folded mitochondrial malate dehydrogenase by GroEL. Steady and time-dependent emission anisotropy measurements.
abstract: The binding of partially-folded mitochondrial malate dehydrogenase (mMDH) to GroEL was assessed by steady and nanosecond emission spectroscopy. Partially-folded intermediates of mMDH show significant residual secondary structure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein-ANS complexes display a rotational correlation time of 19 ns. Similar rotational correlation time (phi = 18.6 ns) was determined for partially-folded species tagged with anthraniloyl. GroEL recognizes partially-folded species with a K(D) approximately 60 nM. The rotational correlation time of the complex, i.e., GroEL-mMDH-ANT, approaches 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 observed rotational correlation time. The results indicate that partially-folded malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conformations endowed with catalytic activity.
authors.affiliation: Department of Biochemistry, University of Tennessee, Knoxville 37996-0840, USA. bcchurch@inet.polyn.edu.hk
authors.forename: J E
authors.initials: JE
authors.lastname: Churchich
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D016880:Anisotropy; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D010649:Phenylalanine; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014443:Tyrosine; D062367:ortho-Aminobenzoates / Q000737:chemistry
chemical_list: D018834:Chaperonin 60; D062367:ortho-Aminobenzoates; D014443:Tyrosine; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D008291:Malate Dehydrogenase