Some structural features of the iron-uptake regulation protein.
An extensive proton nuclear magnetic resonance study of the iron-uptake regulation protein (Fur) from Escherichia coli has been made. Considerable difficulties were experienced in the NMR experiments in 1H2O which may be due unfavourable proton exchange rates in the pH range greater than 6.2, where the protein is soluble. Even in 2H2O, the two-dimensional NMR spectra were not easily interpreted due to widely differing line widths, as a result of the protein side-chains having very differing mobilities. Despite these problems, virtually all the 20 aromatic amino acids have been assigned. Small regions of the protein core were assigned by taking advantage of the approximately 20 non-exchanging peptide-NH resonances in 2H2O. Using two-dimensional J-correlated, homonuclear Hartmann-Hahn and NOE spectroscopies, we have been able to give some assignments in which there is considerable confidence for about one third of the amino acids. Taking advantages of two series of probe experiments, using Mn(II) and a spin label, together with longer range NOE data and result from structure predictions and CD data, we have put forward a tentative fold for the protein which is seen to have a relatively rigid series of interior strands and more flexible exterior strands, many of which are likely to be helical. The Mn(II) probe experiments have also allowed us to define the Fe(II) binding site.
Saito
T
T
Inorganic Chemistry Laboratory, University of Oxford, UK.
Wormald
M R
MR
Williams
R J
RJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003903:Deuterium; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000378:metabolism; D005682:Gadolinium / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014867:Water
D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D014867:Water; D008345:Manganese; D003903:Deuterium; D005682:Gadolinium; D007501:Iron; D017666:Deuterium Oxide