Determination of the conformations of cAMP receptor protein and its T127L,S128A mutant with and without cAMP from small angle neutron scattering measurements
Determination of the conformations of cAMP receptor protein and its T127L,S128A mutant with and without cAMP from small angle neutron scattering measurements
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DOI：
10.1074/jbc.273.32.20001
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发表时间：
1998-08-07
影响因子：
4.8
通讯作者：
Schwarz, FP
中科院分区：
文献类型：
--
作者：
Krueger, S;Gorshkova, I;Schwarz, FP
在不存在和存在3 '，5'环腺苷一磷酸（cAMP）的情况下，对cAMP受体蛋白（CRP）的溶液和CRP的T127 L、S128 A双突变体（CRPQ*）在含有0.5M KCl的D2 O K3 PO 4缓冲液中的溶液进行小角中子散射（SANS）测量。CRP的能量最小化结构通过CRP的X射线晶体学结构的最小化来计算，CRP的X射线晶体学结构为完全“闭合”形式（其中羧基末端结构域的α-螺旋折叠靠近氨基末端结构域）和完全“开放”形式（其中羧基末端结构域的α-螺旋折叠远离氨基末端结构域）。中子散射模型表明，CRP SANS数据与开放形式下未连接的CRP预测的数据曲线密切相关，而cAMP连接的数据与封闭形式下最小化的cAMP连接的CRP结构预测的数据更加一致。因此，看来在与cAMP连接后，溶液中的CRP经历了从开放形式到封闭形式的构象变化。来自CRP* 和cAMP连接的CRP* 的SANS数据是一致的，这意味着两种CRP* 之间的结构差异非常小。这与体内结果一致，体内结果显示CRP仅在cAMP存在下激活细胞中的转录，而CRP* 在cAMP不存在下激活转录，这意味着CRP* 已经处于用于激活转录的正确构象。
Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRPQ*) in D2O K3PO4 buffer containing 0.5 M KCI, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form, Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP*: are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.