Analysis of Receptor-G Protein Interactions by using Receptor Peptide Fragments

通过对G蛋白-受体相互作用的理化分析，我们得到了以下结果，最终目的是阐明G蛋白信号转导的详细机制：G蛋白α亚基的表达：我们建立了一个表达和容易纯化重组Gilalpha的系统。用Ni^<2+>亲和层析法在Gilalpha的n端附着组氨酸标签，可以制备出高达50 mg/L的Gilalpha培养物。该体系还可以得到氘化吉尔。同时制备了一个K349P突变体，该突变体与Gsalpha中的unc突变体相对应。正如预期的那样，这种蛋白不会被受体模拟物激活。建立了Goalpha和Gsalpha的表达系统，但后者有待进一步完善。肽的表达和稳定同位素标记：为了详细分析受体模拟肽与G蛋白之间的相互作用，我们建立了一个系统，将这些肽作为泛素融合蛋白表达，并用稳定同位素（如^<13>C和^<15>N.3）标记它们。Gilalpha结合的mastoparan-X的构象：通过TRNOE分析，在mastoparan-X的Gilalpha存在的情况下，均匀地以^<15>N单独或同时以^<13>C和^<15>N标记，得到了mastoparan-X与Gilalpha结合的精确构象，rmsd小至0.33*。从第三个到c末端的残基呈α -螺旋构象。受体模拟物激活后Gilalpha的构象变化：为了探讨配体受体刺激促进G蛋白GDP释放的机制，用cd分析了受体模拟物引起Gilalpha二级结构的变化，发现Gilalpha的α -螺旋含量随其激活而平行减少。

We obtained the following results by physicochemical analyzes of G protein-receptor interactions which ultimately aim in elucidating detailed mechanisms of signal transduction via G proteins :1.Expression of G protein alpha-subunits : We set up a system to express and easily purify recombinant Gilalpha. As high as 50 mg/L culture of Gilalpha could be prepared by Ni^<2+>-affinity chromatography of Gilalpha by attaching a histidine-tag at the N-terminus. Deuterated Gilalpha could also be obtained by this system. Aslo prepared was a K349P mutant which corresponds to the unc mutant in Gsalpha. As expected, this protein was not activated by receptor mimetics. Expression systems for Goalpha and Gsalpha were setup though the latter needs more refinements.2.Expression and stable isotope labeling of peptides : To analyze interactions between receptor-mimetic peptides and G proteins by NMR in detail, we set up a system that expresses these peptides as a fusion protein with ubiquitin and labels them with stable isotopes such as ^<13>C and ^<15>N.3.Conformation of Gilalpha-bound mastoparan-X : By TRNOE analysis in the presence of Gilalpha of mastoparan-X uniformly labeled with ^<15>N alone or both ^<13>C and ^<15>N,a precise conformation of mastoparan-X bonud to Gilalpha was obtained with rmsd as small as 0.33*. Residues from the third one to the C-terminus was found to take an alpha-helical conformation.4.Conformational change of Gilalpha upon activation by receptor mimetics : To investigate the mechanism where by stimulation by a liganded receptor enhances the GDP release from G protein, secondary structure change of Gilalpha caused by receptor mimetics was analyzed by CD.The alpha-helix content of Gilalpha was found to decrease in parallel with its activation.