Miller A F, Papastavros M Z, Redfield A G
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Biochemistry. 1992 Oct 27;31(42):10208-16. doi: 10.1021/bi00157a007.
1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.
溶液中1H检测的15N编辑核磁共振技术被用于研究人N-p21ras与GDP结合形式和与GTPγS结合形式之间的构象差异。观察到了15N标记的甘氨酸和异亮氨酸的酰胺质子。除了[15N]甘氨酸和[15N]异亮氨酸外，通过掺入各种13C标记的氨基酸以及用Co2+取代Mg2+，将共振信号归属到了特别感兴趣的残基，即甘氨酸-60、-75以及异亮氨酸-21和-36。当GTPγS取代p21ras活性位点中的GDP时，14个甘氨酸酰胺共振信号中只有5个显示出较大的位移，这表明构象效应相当局限。响应的甘氨酸-10、-12、-13和-15位于活性位点。位于构象活性环和螺旋远端的甘氨酸-75也对GTPγS取代GDP作出响应，而甘氨酸-77则没有，这支持了甘氨酸-75作为构象变化旋转点的作用。当GDP被GTPγS取代时，异亮氨酸-36和-21以及第三个未鉴定的异亮氨酸的酰胺质子共振也发生了较大位移。因此，包含异亮氨酸-36的效应物结合环被证实参与了构象变化，并且包含异亮氨酸-21的α-螺旋也显示受到了影响。
