Conformational transitions in p21(ras) and in its complexes with the effector protein Raf-RBD and the GTPase activating protein GAP
Conformational transitions in p21(ras) and in its complexes with the effector protein Raf-RBD and the GTPase activating protein GAP
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DOI：
10.1021/bi952858k
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发表时间：
1996-08-13
期刊：
影响因子：
2.9
通讯作者：
Kalbitzer, HR
中科院分区：
文献类型：
--
作者：
Geyer, M;Schweins, T;Kalbitzer, HR
~（31）P NMR显示p21（ras）与GTP类似物GppNHp的复合物。Mg 2+以两种构象状态存在，状态1和2。在野生型p21（ras）中，两种状态之间的平衡常数K-1（（12））为1.09。不同的突变体，这些菌株的数量不同，但与温度无关。通过全交换矩阵分析确定野生型p21（ras）和p21（ras）（S65 P）的构象转变的活化焓Δ H-双匕首和活化熵Δ S-双匕首。对于野生型蛋白质，得到Δ K-双匕首= 89 +/-2 kJ mol（-1）和Δ S-双匕首= 102 +/-20 J mol（-1）K-1，对于突变蛋白质，得到Δ K-双匕首= 93 +/-7 kJ mol（-1）和Δ S-双匕首= 138 +/-30 J mol（-1）K-1。对各种p21（ras）突变体的研究表明，这两种状态对应于L2环的不同构象，Tyr-32相对于结合核苷酸位于两个不同位置。在95 GHz的高场EPR表明，所观察到的构象转变不直接影响蛋白质结合的金属离子的配位球。用突变体E62 H和E63 H通过H-1 NMR研究了这种转变对L4环的影响。没有迹象表明LA参与了L2中描述的过渡。虽然通过降低pH值可以诱导可逆的构象变化。在核磁共振时间尺度上，两块石板之间的交换是缓慢的（
P-31 NMR revealed that the complex of p21(ras) with the GTP analog GppNHp . Mg2+ exists in two conformational states, states 1 and 2. In wild-type p21(ras) the equilibrium constant K-1((12)) between the two stales is 1.09. The population of these stales is different for various mutants but independent of temperature. The activation enthalpy Delta H-double dagger and activation entropy Delta S-double dagger for the conformational transitions were determined by full-exchange matrix analysis for wild-type p21(ras) and p21(ras)(S65P). For the wildtype protein one obtains Delta K-double dagger = 89 +/- 2 kJ mol(-1) and Delta S-double dagger = 102 +/- 20 J mol(-1) K-1 and for the mutant protein Delta K-double dagger = 93 +/- 7 kJ mol(-1) and Delta S-double dagger = 138 +/- 30 J mol(-1) K-1. The study of various p21(ras) mutants suggests that the two states correspond to different conformations of loop L2, with Tyr-32 in two different positions relative to the bound nucleotide. High-field EPR at 95 GHz suggests that the observed conformational transition does not directly influence the coordination sphere of the protein-bound metal ion. The influence of this transition on loop L4 was studied by H-1 NMR with mutants E62H and E63H. There was no indication that LA takes part in the transition described in L2. although a reversible conformational change could be induced by decreasing the pH value. The exchange between the two slates is slow On the NMR time scale (