期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1834, 期 12, 页码 2512-2519出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2013.08.014
关键词
Flavodoxin; NADP(+) oxidoreductase; Pyruvate formate-lyase activating enzyme; Protein-protein interactions; Surface plasmon resonance (SPR); Circular dichroism (CD)
资金
- NIGMS NIH HHS [2R01GM054608, R01 GM054608] Funding Source: Medline
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. (C) 2013 Elsevier B.V. All rights reserved.
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