Direct visualization of ligand-protein interactions using atomic force microscopy
作者:Calum S Neish、Ian L Martin、Robert M Henderson、J Michael Edwardson
DOI:10.1038/sj.bjp.0704660
日期:2002.4
Streptavidin is a 60‐kDa tetramer which binds four molecules of biotin with extremely high affinity (KA∼1014 M−1). We have used atomic force microscopy (AFM) to visualize this ligand‐protein interaction directly.
Biotin was tagged with a short (152‐basepair; 50‐nm) DNA rod and incubated with streptavidin. The resulting complexes were then imaged by AFM. The molecular volume of streptavidin calculated from the dimensions of the protein particles (105±3 nm3) was in close agreement with the value calculated from its molecular mass (114 nm3). Biotinylation increased the apparent size of streptavidin (to 133±2 nm3), concomitant with an increase in the thermal stability of the tetramer.
Images of streptavidin with one to four molecules of DNA‐biotin bound were obtained. When two ligands were bound, the angle between the DNA rods was either acute or obtuse, as expected from the relative orientations of the biotin binding sites. The ratio of acute : obtuse angles (1 : 3) was lower than the expected value (1 : 2), indicating a degree of steric hindrance in the binding of the DNA‐biotin. The slight under‐representation of higher occupancy states supported this idea.
Streptavidin with a single molecule of DNA‐biotin bound was used to tag biotinylated β‐galactosidase, a model multimeric enzyme.
The ability to image directly the binding of a ligand to its protein target by AFM provides useful information about the nature of the interaction, and about the effect of complex formation on the structure of the protein. Furthermore, the use of DNA‐biotin/streptavidin tags could potentially shed light on the architecture of multi‐subunit proteins.
British Journal of Pharmacology (2002) 135, 1943–1950; doi:10.1038/sj.bjp.0704660
链霉蛋白酶(streptavidin)是一种60千道尔顿(kDa)的四聚体,能够以极高的亲和力结合四个生物素(biotin)分子(结合常数KA约为1014 M−1)。我们利用原子力显微镜(AFM)直接观察了这种配体-蛋白质相互作用。
生物素被标记为一个短的DNA棒(152个碱基对;50纳米),并与链霉蛋白酶孵育。随后,通过AFM对形成的复合物进行了成像。根据蛋白质颗粒的尺寸计算得出链霉蛋白酶的分子体积(105±3 纳米3),与根据其分子质量计算的结果(114 纳米3)非常接近。生物素化处理增加了链霉蛋白酶的表观大小(至133±2 纳米3),同时也提高了四聚体的热稳定性。
得到了链霉蛋白酶与一到四个DNA-生物素分子结合的图像。当两个配体结合时,DNA棒之间的夹角要么是锐角,要么是钝角,这与生物素结合位点的相对取向一致。锐角与钝角的比例(1:3)低于预期值(1:2),表明DNA-生物素结合存在一定程度的空间位阻。高占据态的轻微低表示进一步支持了这一观点。
带有单个DNA-生物素分子结合的链霉蛋白酶被用于标记生物素化的β-半乳糖苷酶(一种模型多聚酶)。
通过AFM直接成像配体与其蛋白质靶标之间的结合,提供了有关相互作用性质的信息,以及复合物形成对蛋白质结构的影响。此外,DNA-生物素/链霉蛋白酶标签的使用可能揭示多亚基蛋白质的结构。
British Journal of Pharmacology (2002)
135, 1943–1950; doi:
10.1038/sj.bjp.0704660