Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range.
Journal article, 2005
A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP(+) or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK(a) values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK(a) values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP(+) and NADPH forms of domain III, but with a marked difference in pK(a) not only attributable to the charge difference between the substrates. Titrating residues found in loop D/alpha5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK(a) values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.
chemistry
Protein Structure
Biomolecular
metabolism
Tertiary
Hydrogen-Ion Concentration
chemistry
Escherichia coli Proteins
NADP
Titrimetry
chemistry
chemistry
NADP Transhydrogenase
Nuclear Magnetic Resonance
Oxidation-Reduction
Protein Subunits
Author
Anders Pedersen
University of Gothenburg
Tomas Johansson
Chalmers, Chemical and Biological Engineering
Jan Rydström
University of Gothenburg
B Göran Karlsson
Chalmers, Chemical and Biological Engineering
Biochimica et biophysica acta
0006-3002 (ISSN)
Vol. 1707 2-3 254-8Subject Categories (SSIF 2011)
Biochemistry and Molecular Biology
DOI
10.1016/j.bbabio.2004.12.004
PubMed
15863102