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首页> 外文期刊>Biochemistry >A Thermodynamic Ligand Binding Study of the Third PDZ Domain (PDZ3) from the Mammalian Neuronal Protein PSD-95
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A Thermodynamic Ligand Binding Study of the Third PDZ Domain (PDZ3) from the Mammalian Neuronal Protein PSD-95

机译：来自哺乳动物神经元蛋白PSD-95的第三个PDZ域（PDZ3）的热力学配体结合研究。

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摘要

The thermodynamic parameters associated with the binding of several series of linear peptides to the third PDZ domain (PDZ3) of the postsynaptic density 95 protein (PSD-95) have been measured using isothermal titration calorimetry (ITC). Two strategies were pursued in developing these binding ligands: (1) systematic N-terminal truncation of sequences derived from the C-terminal regions of identified PDZ3-binding proteins (CRIPT, neuroligin-1, and citron) and (2) selective mutation of specific positions within a consensus hexapeptide (KKETEV) known to bind PDZ3. Each synthetically prepared peptide was used to titrate PDZ3, which yielded the changes in Gibbs free energy (DELTA G), enthalpy (DELTA H), and entropy (T DELTA S) for the binding event. Selected peptides were subjected to additional analysis, which entailed (1) measuring the change in heat capacity (DELTA C_p) upon association, to assess the character of the binding interface, and (2) constructing thermodynamic double mutant cycles, to determine the presence of cooperative effects. From the first series, the CRIPT protein proved to be the better source for higher affinity sequences. From the second series, enhanced binding was associated with peptides that closely adhered to the established motif for class I PDZ domain C-termini, X-(T/S)-X-(V/I/L), and more specifically to a narrower motif of X-T-X-V. Further, in both series a length of six residues was necessary and sufficient to capture maximal affinity. In addition, there were significant influences upon binding by modifying the abutting "X" positions. The cumulative results provide greater detail into the specific nature of ligand binding to PDZ3 and will assist in the development of selective molecular probes for the study of this and structurally homologous PDZ domains.
机译：已经使用等温滴定量热法（ITC）测量了与几个线性肽系列与突触后密度95蛋白（PSD-95）的第三个PDZ域（PDZ3）结合相关的热力学参数。在开发这些结合配体时，采取了两种策略：（1）系统性地从鉴定出的PDZ3结合蛋白（CRIPT，neuroligin-1和citron）的C端区域衍生的序列的N端截短，以及（2）选择性突变已知结合PDZ3的共有六肽（KKETEV）中的特定位置。每种合成制备的肽均用于滴定PDZ3，从而产生结合事件的吉布斯自由能（DELTA G），焓（DELTA H）和熵（T DELTA S）变化。对选定的肽进行进一步分析，这需要（1）缔合后测量热容的变化（DELTA C_p），以评估结合界面的特征，以及（2）构建热力学双突变循环，以确定是否存在合作效应。从第一个系列开始，CRIPT蛋白被证明是更高亲和力序列的更好来源。在第二个系列中，增强的结合与紧密粘附到I类PDZ域C-末端，X-（T / S）-X-（V / I / L）的已确定基序的肽相关，更具体地讲，与XTXV的较窄主题。此外，在两个系列中，六个残基的长度都是必需的，并且足以捕获最大的亲和力。另外，通过修饰邻接的“ X”位置对结合有显着影响。累积的结果为配体与PDZ3结合的特定性质提供了更多细节，并将有助于开发选择性分子探针以研究该PDZ和结构同源的PDZ域。

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