Secondary structural analysis of the Na(+),K(+)-ATPase and its Na(+) (E(1)) and K(+) (E(2)) complexes by FTIR spectroscopy.
The Na(+),K(+)-ATPase is an integral membrane protein which transports sodium and potassium cations against an electrochemical gradient. The transport of Na(+) and K(+) ions is presumably connected to an oscillation of the enzyme between the two conformational states, the E(1) (Na(+)) and the E(2) (K(+)) conformations. The E(1) and E(2) states have different affinities for ligand interaction. However, the determination of the secondary structure of this enzyme in its sodium and potassium forms has been the subject of much controversy. This study was designed to provide a quantitative analysis of the secondary structure of the Na(+),K(+)-ATPase in its sodium (E(1)) and potassium (E(2)) states in both H(2)O and D(2)O solutions at physiological pH, using Fourier transform infrared (FTIR) with its self-deconvolution and second derivative resolution enhancement methods, as well as curve-fitting procedures. Spectroscopic analysis showed that the secondary structure of the sodium salt of the Na(+),K(+)-ATPase in H(2)O solution contains alpha-helix 19.8+/-1%, beta-sheet 25.6+/-1%, turn 9.1+/-1%, and beta-anti 7.5+/-1%, whereas in D(2)O solution, the enzyme shows alpha-helix 16.8+/-1%, beta-sheet 24.5+/-1.5%, turn 10.9+/-1%, beta-anti 9.8+/-1%, and random coil 38.0+/-2%. Similarly, the potassium salt in H(2)O solution contains alpha-helix 16.6+/-1%, beta-sheet 26.4+/-1.5%, turn 8.9+/-1%, and beta-anti 8.1+/-1%, while in D(2)O solution it shows alpha-helix 16.2+/-1%, beta-sheet 24.5+/-1.5%, turn 10.3+/-1%, beta-anti 9.0+/-1%, and random coil 40+/-2%. Thus the main differences for the sodium and potassium forms of the Na(+),K(+)-ATPase are alpha-helix 3.2% in H(2)O and 0.6% in D(2)O, beta-sheet (pleated and anti) 1.5% in H(2)O and random structure 2% (D(2)O), while for other minor components (turn structure), the differences are less than 1%.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D011188:Potassium / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012964:Sodium / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared
D012996:Solutions; D012964:Sodium; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium