Using ANS to Probe Ligand Induced Conformational States of Malate Dehydrogenase
In: The FASEB Journal ; volume 30, issue S1 ; ISSN 0892-6638 1530-6860, 2016
academicJournal
Zugriff:
Malate Dehydrogenase catalyzes the oxidation/reduction of malate/oxaloacetate coupled with the conversion of NAD and NADH and plays a critical role in energy metabolism in the cell. The enzyme is a dimer and has been suggested to involve subunit interactions in both its normal function and in regulation by citrate. 8‐Anilinonaphthalene‐1‐sulfonic acid (ANS) is frequently used as a conformational probe of protein structure since it often shows a dramatic increase in fluorescence and spectral shifts on binding to exposed regions of a protein and has been used to follow protein folding, ligand induced conformational changes, and aggregation. However little is known about the specifics of changes in fluorescence resulting from different modes of binding. To address this issue, and to characterize ligand induced conformational states in Malate Dehydrogenase we have used a combination of spectral studies combined with ANS titrations to characterize the different modes of binding of ANS to Malate Dehydrogenase in the presence or absence of a variety of ligands. Titrations reveal both high and low affinity sites on the protein. Computational docking studies with both “open” and “closed” conformations shown to exist by x ray crystallography, have been used to identify potential regions of the protein which may constitute these sites. The spectral properties of the various sites are resolved using “Peakfit” to deconvolute the experimental data. Using a titration and spectral deconvolution approach we have examined mitochondrial, cytosolic and glyoxysomal forms of the enzyme to elucidate the effects of addition of varying concentrations of the substrates, Malate and Oxaloacetate, as well as several inhibitors of the enzyme, succinate and malonate on the conformational states of the enzyme. The results suggest a complex array of conformational states associated with possible spatial asymmetry and ligand induced changes in the protein.
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Using ANS to Probe Ligand Induced Conformational States of Malate Dehydrogenase
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Autor/in / Beteiligte Person: | Schwabe, Michael ; Bell, Ellis ; Bell, Jessica |
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Zeitschrift: | The FASEB Journal ; volume 30, issue S1 ; ISSN 0892-6638 1530-6860, 2016 |
Veröffentlichung: | Wiley, 2016 |
Medientyp: | academicJournal |
DOI: | 10.1096/fasebj.30.1_supplement.600.19 |
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