Electron paramagnetic resonance and fluorescence studies of the conformation of aspartate aminotransferase bound to GroEL.
The interaction of the precursor to mitochondrial aspartate aminotransferase (pmAAT) with GroEL has been studied by electron paramagnetic resonance (EPR) and fluorescence spectroscopy. In the native protein, the spin probe was immobilized when attached to Cys166 at the domain interface, but was fully mobile when introduced at Cys(-19) in the N-terminal presequence peptide. Unfolding of the protein resulted in a highly mobile EPR spectrum for probes introduced at either site. However, the nitroxide group in GroEL-bound pmAAT showed either intermediate or high mobility depending on the spin probe used. Power saturation experiments indicated that the accessibility of the nitroxide side chain to Ni(EDDA) in the GroEL-pmAAT complex was higher than in the native state when in position 166 but lower when at position -19. Similar results were obtained in fluorescence quenching experiments. These data suggest that GroEL binds partly folded states of pmAAT with the presequence peptide probably in direct contact with GroEL. GroES and ATP, but not AMP-PNP or ADP, support refolding of pmAAT. During refolding, the rate of recovery of the native spectroscopic properties of labeled Cys166 is nearly identical to the rate-limiting reactivation step. Thus, correct docking of the large and small domains of pmAAT may be a key structural event in the regain of catalytic activity.
Berezov
Alan
A
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA.
McNeill
Megan J
MJ
Iriarte
Ana
A
Martinez-Carrion
Marino
M
D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013113:Spin Labels
D018834:Chaperonin 60; D005456:Fluorescent Dyes; D012997:Solvents; D013113:Spin Labels; D001219:Aspartate Aminotransferases