Determination of affinities for lck SH2 binding peptides using a sensitive fluorescence assay: Comparison between the pYEEIP and pYQPQP consensus sequences reveals context-dependent binding specificity
Determination of affinities for lck SH2 binding peptides using a sensitive fluorescence assay: Comparison between the pYEEIP and pYQPQP consensus sequences reveals context-dependent binding specificity
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DOI：
10.1021/bi9620868
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发表时间：
1996-12-24
期刊：
影响因子：
2.9
通讯作者：
Grygon, CA
中科院分区：
文献类型：
--
作者：
CousinsWasti, RC;Ingraham, RH;Grygon, CA
本文描述了一种用于评价磷酸酪氨酸（pY）肽与溶液中lck的重组SH2结构域结合的灵敏荧光结合试验的发展。对几种含有病毒性仓鼠多瘤中间T抗原（pYEEI）一致序列的荧光肽进行了鉴定。这些肽含有乙酰胺-苯胺-萘磺酸（AANS）、丙烯酰丹或丹基作为荧光团。这些探针的光谱特征是在存在和不存在lack SH2结构域的情况下表征的。所研究的荧光肽的结合亲和度（Kd）在40 ~ 500 nM之间。含有FTATEC(AANS)QpYEEIP序列的荧光肽在结合SH2结构域后显示出最高的结合亲和力（K-d 3.98 × 10(-8) M）和最大的发射强度变化（约8.7倍）。该探针随后被用于竞争性结合实验，以研究缺乏SH2结构域与pYEEIP和pYQPQP （pY(505) c端）一致序列相关的一系列磷酸肽的相互作用。从结合亲和的角度讨论了pYEEIP序列中肽长度和残基取代的影响。两个肽系列的比较表明，个体取代对结合亲和力的贡献是依赖于上下文的。这些数据还得出结论，P在+2处的存在会导致结合序列的功能性“截断”；这种隐式截断实际上可能是pYQPQP序列的自调节性质所期望的特性，因为它保留了SH2结构域的特异性，同时将Kd调节到适当的值，以维持信号转导事件中涉及的受体-配体相互作用的微妙平衡。
The development of a sensitive fluorescence binding assay for evaluating the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides containing the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the acetamido-anilino-naphthyl sulfonic acid (AANS), acrylodan, or dansyl groups as fluorophores. The spectral features of these probes were characterized in the presence and absence of the lck SH2 domain. The binding affinities (Kd) for the fluorescent peptides studied ranged from 40 to 500 nM. The fluorescent peptide containing the sequence FTATEC(AANS)QpYEEIP exhibited the highest binding affinity (K-d 3.98 x 10(-8) M) and largest change in emission intensity (approximate to 8.7-fold) upon binding the SH2 domain. This probe was subsequently used in competitive binding assays to study the interaction of the lck SH2 domain with a series of phosphopeptides related to the pYEEIP and pYQPQP (the pY(505) C-terminal) consensus sequences. The effects of peptide length and substitutions of residues within the pYEEIP sequence are discussed in terms of binding affinities. Comparison between the two peptide series revealed that the contributions of individual substitutions to binding affinity are context-dependent. The data also led to the conclusion that the presence of P at +2 results in a functional ''truncation'' of the binding sequence; i.e., residues at positions higher than +2 do not participate significantly in binding, This implicit truncation may actually be a desired property for the autoregulatory nature of the pYQPQP sequence, since it retains specificity for the SH2, domain while adjusting the Kd to a value appropriate for maintaining the delicate balance of receptor-ligand interactions that are involved in signal transduction events.