Farrow N A, Muhandiram R, Singer A U, Pascal S M, Kay C M, Gish G, Shoelson S E, Pawson T, Forman-Kay J D, Kay L E
Protein Engineering Network Centres of Excellence, University of Toronto, Ontario, Canada.
Biochemistry. 1994 May 17;33(19):5984-6003. doi: 10.1021/bi00185a040.
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 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), 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 and 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 may 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-to-millisecond-time scale.
对磷脂酶Cγ1 C末端SH2结构域的主链动力学进行了研究。研究了该结构域的两种形式，一种与源自血小板衍生生长因子受体的高亲和力结合肽形成复合物，另一种则不存在该肽。采用脉冲场梯度的二维1H-15N NMR方法来测定稳态1H-15N NOE值以及T1和T2 15N弛豫时间。主链动力学通过整体相关时间（τm）、序参数（S2）、内部运动的有效相关时间（τe）来表征，如有需要，还包括用于解释微秒至毫秒时间尺度上运动的项。对于具有弛豫数据且无法用单时间尺度形式主义充分拟合的残基，采用了扩展的双时间尺度形式主义。发现未复合和复合形式的SH2的整体相关时间分别为9.2和6.5 ns，这表明未复合形式处于单体 - 二聚体平衡状态。随后通过流体动力学测量证实了这一点。序参数分析表明，在两种形式的SH2中，所谓的磷酸酪氨酸结合环中的残基均表现出高于平均水平的无序性。尽管在未复合和复合形式的SH2之间观察到序参数存在局部差异，但总体而言，在肽结合形式中未发现更高的序参数，这表明平均而言，结合肽后皮秒时间尺度上的无序性并未降低。与未复合形式相比，SH2 - 磷酸肽复合物的弛豫数据用更少的交换项就能拟合。这可能反映了未复合形式中存在的单体 - 二聚体平衡，或者可能表明复合形式在微秒至毫秒时间尺度上具有较低的构象灵活性。
