Cores and pH-dependent dynamics of ferredoxin-NADP+ reductase revealed by hydrogen/deuterium exchange

J Biol Chem. 2007 Feb 23;282(8):5959-67. doi: 10.1074/jbc.M608417200. Epub 2006 Dec 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chloroplasts / enzymology*
  • Deuterium / chemistry*
  • Ferredoxin-NADP Reductase / chemistry*
  • Ferredoxin-NADP Reductase / metabolism
  • Ferredoxins / chemistry
  • Ferredoxins / metabolism
  • NADP / chemistry
  • NADP / metabolism
  • Nuclear Magnetic Resonance, Biomolecular / methods
  • Plant Proteins / chemistry*
  • Plant Proteins / metabolism
  • Protein Folding*
  • Structure-Activity Relationship
  • Zea mays / enzymology*

Substances

  • Ferredoxins
  • Plant Proteins
  • NADP
  • Deuterium
  • Ferredoxin-NADP Reductase