Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

Abstract

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residuebased conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP ؉ reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP ؉ )-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD r values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD r values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP ؉ -binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP ؉ -binding domain at pD r 8.0, the daytime pH in chloroplasts, than at pD r 6.0 is likely to be involved in the increased binding of NADP ؉ for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.Protein conformations as shown by x-ray crystallography or nuclear magnetic resonance spectroscopy are virtually dynamic entities over various time ranges in solution (1, 2). Clarifying such conformational motions at the level of the atom or residue is essential for understanding the structural stabilities of proteins and the relationships between protein structures and functions (3-5). The hydrogen/deuterium (H/D) 4 exchange of amide protons in backbones has become an important way to address the motions of a protein from small or relatively large scale motions involved in biological functions such as binding of a substrate or releasing a product to much more large scale motions like a global unfolding process (4, 6). Among several approaches, the H/D exchange combined with heteronuclear NMR spectroscopy is the most convenient and powerful way because it can provide residue-specific information for most residues (7). For many globular proteins, this approach has identified protected cores, which are often composed of secondary structures buried inside the molecules and the cooperative interactions with partner molecules (4, 8), leading to allostery (9). Detailed analyses of exchanges in the native state of cytochrome c in the presence of various concentrations of denaturants suggested a pathway to unfolding, in which groups of secondary structural elements (i.e. foldons) are unfolded sequentially through distin...

Physicochemical nature of interfaces controlling ferredoxin NADP+ reductase activity through its interprotein interactions with ferredoxin

“…The preparation of recombinant maize leaf FNR (L-FNR I) and 15 N-labeled FNR was based on previous studies [19,30]. Fd and FNR concentrations were determined using the molar extinction coefficients of 9680 M −1 cm −1 at 423 nm and 10,000 M −1 cm −1 at 460 nm, respectively.…”

ArabidopsisTic62 and Ferredoxin-NADP(H) Oxidoreductase Form Light-Regulated Complexes That Are Integrated into the Chloroplast Redox Poise

“…Chemical shift mapping by nuclear magnetic resonance (NMR) spectroscopy has been successfully used to study proteinprotein interactions of large flavoprotein complexes (Maeda et al, 2005;Lee et al, 2007), which is why we applied this method to investigate the interaction between FNR and Tic62. Changes in the peak positions (resonances) or in the signal intensities after titration of a ligand molecule are referred to as chemical shift perturbations, which are suggested to correspond to the interaction sites with the other molecule.…”

“…It is therefore quite reasonable to suggest that the soluble pool of FNR in the stroma is the most responsible for the photosynthetic electron transport and that binding to the thylakoids might serve a different purpose, possibly in redox sensing or regulation. In addition, since FNR stability seems to be lowered at more acidic pH values (Lee et al, 2007), assembly into Tic62 complexes could therefore stabilize the enzyme during the hour of (photosynthetic) inactivity.…”

“…Samples of uniformly 2 H/ 15 N-labeled maize (Zea mays) FNR were prepared as described previously (Maeda et al, 2005;Lee et al, 2007), and the R1 peptide (amino acid sequence: VAKTEQPLSPYTAYDDL-KPPSSPSPTKPSE) was purchased from Toray Research Center. The NMR samples were dissolved in 25 mM sodium phosphate buffer, pH 8.0, 50 mM NaCl, and 90% H 2 O/10% D 2 O.…”

Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents

“…Similarly, in vitro alkalization of isolated thylakoids dissociated FNR and Tic62 (Benz et al, 2009). It is important to stess that the interaction of Tic62 with FNR stabilizes the activity of the FNR protein (Benz et al, 2009) and that FNR activity is lower in acidic than basic environment (Lee et al, 2007). These results indicate that Tic62 acts as a chaperone for FNR, and protects the flavoenzyme from inactivation and degradation during the photosynthetically inactive periods, e.g.…”