Ciubotaru Mihai, Ptaszek Leon M, Baker Gary A, Baker Sheila N, Bright Frank V, Schatz David G
Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, New Haven, Connecticut 06510, USA.
J Biol Chem. 2003 Feb 21;278(8):5584-96. doi: 10.1074/jbc.M209758200. Epub 2002 Dec 17.
The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K(D) = 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.
RAG1和RAG2蛋白共同构成了一种核酸酶，该核酸酶在一种称为V(D)J重组的反应中启动免疫球蛋白和T细胞受体基因的组装。RAG1在RAG1/2复合物对重组信号序列（RSS）的识别中起核心作用。为了研究控制RAG1-RSS相互作用的参数，已从细菌中纯化出与短链霉亲和标签融合的小鼠核心RAG1蛋白（氨基酸377 - 1008），使其达到同质状态。在不存在DNA的情况下，链霉亲和标签-RAG1（StrRAG1）蛋白在很宽的蛋白质浓度范围（25 - 500 nM）内以二聚体形式存在，并以相当高的亲和力和特异性（表观K(D) = 41 nM）与RSS结合。电泳迁移率变动分析和偏振各向异性实验均表明溶液中仅存在单一的StrRAG1-DNA复合物。通过频域光谱法测量的各向异性衰减表明该复合物包含一个与单个DNA分子结合的StrRAG1二聚体。利用蛋白质固有荧光和圆二色性测量，我们证明StrRAG1在结合RSS后会发生重大构象变化。稳态荧光和丙烯酰胺猝灭研究表明，这种构象变化与蛋白质固有荧光团从疏水环境重新定位到溶剂暴露环境有关。RSS诱导的StrRAG1构象变化可能会影响RAG1与RAG2的相互作用以及突触复合物的形成。
