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Conformational modulation and hydrodynamic radii of CP 12 protein and its complexes probed by fluorescence correlation spectroscopy
Author(s) -
Moparthi Satish Babu,
ThieulinPardo Gabriel,
Mansuelle Pascal,
Rigneault Hervé,
Gontero Brigitte,
Wenger Jérôme
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12854
Subject(s) - chlamydomonas reinhardtii , fluorescence correlation spectroscopy , glyceraldehyde 3 phosphate dehydrogenase , chemistry , mutant , hydrodynamic radius , biophysics , fluorescence spectroscopy , crystallography , dehydrogenase , fluorescence , biology , biochemistry , enzyme , physics , molecule , organic chemistry , quantum mechanics , aqueous solution , micelle , gene
Light/dark regulation of the Calvin cycle in oxygenic photosynthetic organisms involves the formation and dissociation of supramolecular complexes between CP 12, a nuclear‐encoded chloroplast protein, and the two enzymes glyceraldehyde‐3‐phosphate dehydrogenase ( GAPDH ) ( EC 1.2.1.13 ) and phosphoribulokinase ( PRK ) ( EC 2.7.1.19 ). Despite the high importance of understanding the structural basis of the interaction of CP 12 with GAPDH and PRK to investigate the regulation of the Calvin cycle, information is still lacking about the structural remodulation of CP 12 and its complex formation. Here, we characterize the diffusion dynamics and hydrodynamic radii of CP 12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments. We quantify a hydrodynamic radius of 3.4 ± 0.2 nm for the CP 12 protein with an increase up to 5.2 ± 0.3 nm upon complex formation with GAPDH and PRK . In addition, unfolding experiments reveal a 1.6‐ and 2.0‐fold increase respectively of the hydrodynamic radii for the N‐terminal and C‐terminal cysteine CP 12 mutant proteins compared with their native folded structures. The different behavior of the CP 12 mutant proteins during hydrophobic collapse transition is a direct clue to different structural orientations of the CP 12 mutant proteins. These different structures are expected to facilitate the binding of either GAPDH or PRK during binary complex and ternary complex formation. Structured digital abstractGAPDH , CP12 and PRK physically interact by fluorescence correlation spectroscopy ( View interaction ) CP12 and PRK bind by fluorescence correlation spectroscopy ( View interaction ) GAPDH and CP12 bind by fluorescence correlation spectroscopy ( View interaction )