tailieunhanh - Báo cáo khoa học: Solution structure of the active-centre mutant I14A of the histidinecontaining phosphocarrier protein from Staphylococcus carnosus

High-pressure NMR experiments performed on the histi-dine-containing phosphocarrier protein (HPr) fromSta-phylococcus carnosushave shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy. . | Eur. J. Biochem. 271 4815-4824 2004 FEBS 2004 doi Solution structure of the active-centre mutant I14A of the histidine-containing phosphocarrier protein from Staphylococcus carnosus Andreas Moglich1 Brigitte Koch2 Wolfram Gronwald1 Wolfgang Hengstenberg2 Eike Brunner1 and Hans Robert Kalbitzer1 1 Institute of Biophysics and Physical Biochemistry University of Regensburg Germany 2SG Physiology of Microorganisms Ruhr-University of Bochum Germany High-pressure NMR experiments performed on the histi-dine-containing phosphocarrier protein HPr from Staphylococcus carnosus have shown that residue Ile14 which is located in the active-centre loop exhibits a peculiarly small pressure response. In contrast the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue the mutant protein HPr I14A in which Ile14 is replaced by Ala was produced and studied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds 49 1Hn-15N and 25 1HN-1H residual dipolar couplings a well resolved three-dimensional structure could be determined. The overall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a dis placement of the ring system of His15 and a distortion of the N-terminus of the first helix which supports the histidine ring. In addition the C-terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon which is known to occupy hydrophobic cavities causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phosphocarrier activity of the mutant and agree well with the earlier suggestion that Ile14 represents an anchoring point stabilizing the .