Gibson A L, Herron J N, He X M, Patrick V A, Mason M L, Lin J N, Kranz D M, Voss E W, Edmundson A B
Department of Biology, University of Utah, Salt Lake City 84112.
Proteins. 1988;3(3):155-60. doi: 10.1002/prot.340030304.
An antigen-binding fragment (Fab) from a murine monoclonal antibody (4-4-20) with high affinity for fluorescein was cocrystallized with ligand in polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) in forms suitable for X-ray analyses. In MPD the affinity of the intact antibody for fluorescein was 300 times lower than the value (3.4 x 10(10) M-1) obtained in aqueous buffers. This decreased affinity was manifested by the partial release of bound fluorescein when MPD was added to solutions of liganded Fab during crystallization trials. In PEG, the ligand remained firmly bound to the protein. The liganded Fab crystallized in the monoclinic space group P2(1) in PEG, with a = 58.6, b = 97.2, c = 44.5 A and beta = 95.2 degrees. In MPD the space group was triclinic P1, with a = 58.3, b = 43.4, c = 42.3 A, alpha = 83.9 degrees, beta = 87.6 degrees, and gamma = 84.5 degrees. X-ray diffraction data were collected for both forms to 2.5-A resolution. Surprisingly, the triclinic form of the liganed antifluorescyl Fab had the same space group, closely similar cell dimensions, and practically the same orientation in the unit cell as an unliganded Fab (BV04-01) with activity against single-stranded DNA.
一种对荧光素有高亲和力的鼠单克隆抗体（4-4-20）的抗原结合片段（Fab）与配体在聚乙二醇（PEG）和2-甲基-2,4-戊二醇（MPD）中共同结晶，形成适合X射线分析的形式。在MPD中，完整抗体对荧光素的亲和力比在水性缓冲液中获得的值（3.4×10¹⁰ M⁻¹）低300倍。这种亲和力的降低表现为在结晶试验期间将MPD添加到结合荧光素的Fab溶液中时，结合的荧光素会部分释放。在PEG中，配体与蛋白质保持牢固结合。结合配体的Fab在PEG中以单斜空间群P2(1)结晶，a = 58.6，b = 97.2，c = 44.5 Å，β = 95.2°。在MPD中，空间群为三斜P1，a = 58.3，b = 43.4，c = 42.3 Å，α = 83.9°，β = 87.6°，γ = 84.5°。收集了两种形式的X射线衍射数据，分辨率达到2.5 Å。令人惊讶的是，结合配体的抗荧光素Fab的三斜形式与具有抗单链DNA活性的未结合配体的Fab（BV04-01）具有相同的空间群、非常相似的晶胞尺寸以及在晶胞中几乎相同的取向。
Zotero 插件