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Recognition of partially‐folded mitochondrial malate dehydrogenase by GroEL. Steady and time‐dependent emission anisotropy measurements
Author(s) -
Churchich Jorge E.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560071212
Subject(s) - groel , malate dehydrogenase , rotational correlation time , nanosecond , chaperonin , biophysics , chemistry , crystallography , biochemistry , biology , enzyme , protein folding , physics , molecule , escherichia coli , laser , organic chemistry , optics , gene
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 (ø = 18.6 ns) was determined for partially‐folded species tagged with anthraniloyl. GroEL recognizes partially‐folded species with a K D ⋍ 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.