title: Flavodoxin cofactor binding induces structural changes that are required for protein-protein interactions with NADP(+) oxidoreductase and pyruvate formate-lyase activating enzyme.
abstract: Flavodoxin (Fld) conformational changes, thermal stability, and cofactor binding were studied using circular dichroism (CD), isothermal titration calorimetry (ITC), and limited proteolysis. Thermodynamics of apo and holo-Fld folding were examined to discern the features of this important electron transfer protein and to provide data on apo-Fld. With the exception of fluorescence and UV-vis binding experiments with its cofactor flavin mononucleotide (FMN), apo-Fld is almost completely uncharacterized in Escherichia coli. Fld is more structured when the FMN cofactor is bound; the association is tight and driven by enthalpy of binding. Surface plasmon resonance binding experiments were carried out under anaerobic conditions for both apo- and holo-Fld and demonstrate the importance of structure and conformation for the interaction with binding partners. Holo-Fld is capable of associating with NADP(+)-dependent flavodoxin oxidoreductase (FNR) and pyruvate formate-lyase activating enzyme (PFL-AE) whereas there is no detectable interaction between apo-Fld and either protein. Limited proteolysis experiments were analyzed by LC-MS to identify the regions in Fld that are involved in conformation changes upon cofactor binding. Docking software was used to model the Fld/PFL-AE complex to understand the interactions between these two proteins and gain insight into electron transfer reactions from Fld to PFL-AE.
authors.affiliation: Department of Chemistry & Biochemistry and the Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Adam V
authors.initials: AV
authors.lastname: Crain
authors.forename: Joan B
authors.initials: JB
authors.lastname: Broderick
mesh_terms: D000123:Acetyltransferases; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D062105:Molecular Docking Simulation; D011485:Protein Binding; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes; D029968:Escherichia coli Proteins; D005418:Flavodoxin; D005420:Flavoproteins; C120194:FldA protein, Bacteria; C077565:fpr protein, E coli; D005287:Ferredoxin-NADP Reductase; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme
keywords: Circular dichroism (CD); Flavodoxin; NADP(+) oxidoreductase; Protein–protein interactions; Pyruvate formate-lyase activating enzyme; Surface plasmon resonance (SPR)
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