In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display
In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display
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DOI：
10.1021/bi0498398
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发表时间：
2004-07-20
期刊：
影响因子：
2.9
通讯作者：
Roberts, RW
中科院分区：
文献类型：
--
作者：
Ja, WW;Roberts, RW
G蛋白调节（GPR）基序类似于20个残基的保守结构域，充当G（i/o α）亚基的鸟嘌呤解离抑制剂（GDI）。在这里，我们描述了分离的肽来自GPR的共识序列使用mRNA展示选择库。生物素化的G（ialpha 1），在N或C末端修饰，作为mRNA展示的GPR肽的高亲和力结合靶点。基于GPR基序的C末端，使用mRNA展示文库进行体外选择，发现具有保守残基的新肽序列。令人惊讶的是，所选的肽含有GPR基序中高度保守的Arg的突变，先前显示出对结合和抑制活性至关重要。来自选择的优势肽R6 A和最小的9聚体肽R6 A-1不含Arg残基，但保留对G（ialpha 1）的GDP结合状态的高亲和力（K-D分别为60和200 nM）和特异性，如通过表面等离子体共振测量的。所选肽还保持G（ialpha 1）的GDI活性，抑制GTP γ S结合测定中的GDP交换和AlF 4刺激的内源性色氨酸荧光增强。然而，GDI活性的动力学对于所选择的肽是不同的，并且表现出双相动力学，表明了复杂的抑制机制。与GPR基序一样，R6 A和R6 A-1肽与G（β-γ）亚基竞争结合G（α 1），表明它们可用作G（β-γ）信号传导的激活剂。
The G protein regulatory (GPR) motif is a similar to20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G(i/oalpha) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G(ialpha1), modified at either the N or C terminus, serves as a high-affinity binding target for mRNA-displayed GPR peptides. In vitro selection using mRNA display libraries based on the C terminus of the GPR motif revealed novel peptide sequences with conserved residues. Surprisingly, selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A, and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K-D = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(ialpha1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G(ialpha1), inhibiting both the exchange of GDP in GTPgammaS binding assays and the AlF4--stimulated enhancement of intrinsic tryptophan fluorescence. The kinetics of GDI activity, however, are different for the selected peptides and demonstrate biphasic kinetics, suggesting a complex mechanism for inhibition. Like the GPR motif, the R6A and R6A-1 peptides compete with G(betagamma) subunits for binding to G(ialpha1), suggesting their use as activators of G(betagamma) signaling.