Abstract
NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based 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.
Highlights
IntroductionThe hydrogen/deuterium (H/D) 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]
Teins and the relationships between protein structures and functions [3,4,5]
The present study showed that the dynamics of the NADPϩ-flexible subdomain, of the four ␣-helices, increased in response to a pDr change from 6.0 to 8.0, suggesting that this elevated dynamics is responsible for the enhanced affinity of ferredoxin-NADP؉ reductase (FNR) for NADPϩ
Summary
The hydrogen/deuterium (H/D) 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]. Ferredoxin-NADPϩ reductase (FNR, 1.18.1.2) from maize leaf is a ubiquitous protein consisting of 314 amino acid residues. We have been studying the conformation and function of maize leaf FNR through heteronuclear NMR analyses with the recently achieved assignment for more than 95% of its amide protons [20]. The results revealed the three protected core regions of FNR in which the NADPϩ-binding domain can be divided into two subdomains with distinct differences in their motions. Comparison of the apparent free energy change of unfolding (⌬GHX) estimated for each residue on the basis of H/D exchange with the global free energy change of unfolding (⌬GU) estimated from urea-induced unfolding suggests that H/D exchange provides a more reliable estimate of the stability of the core regions
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
