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首页> 外文期刊>The FEBS journal >Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTP gamma S
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Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTP gamma S

机译：鸟嘌呤核苷酸结合蛋白Ras与GTP类似物GTPγS复合的慢构象动力学

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摘要

The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K-12 of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido) triphosphate or guanosine-5'-(b,c-methyleno) triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state ( state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) leads to P-31 NMR spectra that indicate the prevalence of only one conformational state with K-12 > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTP gamma S complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affnity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy Delta H-1a1b(vertical bar) of 61 kJ(.)mol(-1) and an activation entropy Delta S-1a1b(vertical bar) of 65 J(.)K(-1.)mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K-A for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTP gamma S complex.
机译：当GTP类似物鸟苷5'-（β，γ-亚氨基）三磷酸或鸟苷时，鸟嘌呤核苷酸结合蛋白Ras以两种不同的构象状态（状态1和状态2）在溶液中出现，平衡常数K-12为2.0。 -5'-（b，c-甲基烯基）三磷酸键合到活性中心。假设状态2代表效应子的强结合状态，其构象与复合到效应子上的Ras相似。在另一种状态（状态1）中，Ras的开关区域很可能是动态无序的。主要存在于状态1中的Ras变体与效应子的亲和力大大降低。相反，与GTP类似物鸟苷5'-O-（3-硫代三磷酸）（GTPγS）结合的Ras（wt）导致P-31 NMR光谱，表明K-12> 10.用Raf激酶的Ras结合域（Raf-RBD）滴定表明该状态对应于效应子结合状态2。在效应子环突变体Ras（T35S）和Ras（T35A）的GTPγS复合物中，两个检测到与状态2不同的构象状态，它们在毫秒的时间尺度上相互转换。与Raf-RBD的结合研究表明，这两个突变体主要存在于低亲和力状态1a和1b中。通过对在不同温度下测得的光谱进行线形分析，可以得出61 kJ（。）mol（-1）的活化能Delta H-1a1b（垂直线）和65 J（垂直线）的活化熵Delta S-1a1b（垂直线） 。）K（-1。）mol（-1）得到。等温滴定热量法结合到不同的GTP类似物上显示，与野生型相比，Raf-RBD与Ras（T35S）的有效亲和力KA降低了约20倍，其中GTP的降低最强γS复合物。

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