BACKBONE DYNAMICS OF A FREE AND A PHOSPHOPEPTIDE-COMPLEXED SRC HOMOLOGY-2 DOMAIN STUDIED BY N-15 NMR RELAXATION
BACKBONE DYNAMICS OF A FREE AND A PHOSPHOPEPTIDE-COMPLEXED SRC HOMOLOGY-2 DOMAIN STUDIED BY N-15 NMR RELAXATION
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DOI：
10.1021/bi00185a040
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发表时间：
1994-05-17
期刊：
影响因子：
2.9
通讯作者：
KAY, LE
中科院分区：
文献类型：
--
作者：
FARROW, NA;MUHANDIRAM, R;KAY, LE
磷脂酶C γ 1的C-末端SH 2结构域的骨架动力学进行了研究。研究了两种形式的结构域，一种是与来自血小板衍生生长因子受体的高亲和力结合肽复合，另一种是在不存在这种肽的情况下。2-D H-1-N-15 NMR方法，采用脉冲场梯度，用于确定稳态H-1-N-15 NOE值和T-1和T-2 N-15弛豫时间。骨架动力学的特征在于整体相关时间（τ（m）），顺序参数（S-2），内部运动的有效相关时间（τ（e）），如果需要的话，在微秒到毫秒的时间尺度上的运动。一个扩展的两个时间尺度的形式主义被用于残留物的松弛数据，不能充分使用一个单一的时间尺度的形式主义。SH 2的未络合和络合形式的总体相关时间分别为9.2和6.5 ns，表明未络合形式处于单体-二聚体平衡。这随后通过流体动力学测量得到证实。序参数的分析表明，在所谓的磷酸酪氨酸结合环中的残基表现出高于平均水平的两种形式的SH 2的障碍。尽管在未复合和复合形式的SH 2之间观察到有序参数的局部差异，但总体而言，在肽结合形式中未发现更高阶的参数，表明平均而言，结合肽后皮秒时间尺度的紊乱没有减少。SH 2-磷酸肽复合物的弛豫数据与未复合形式相比具有更少的交换项。这反映了以未复合形式存在的单体-二聚体平衡，或者可能表明复合形式在微秒到毫秒的时间尺度上具有较低的构象灵活性。
The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D H-1-N-15 NMR methods, employing pulsed field gradients, were used to determine steady-state H-1-N-15 NOE values and T-1 and T-2 N-15 relaxation times. Backbone dynamics were characterized by the overall correlation time (tau(m)), Order parameters (S-2), effective correlation times for internal motions (tau(e)), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This mat reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-tomillisecond-time scale.