Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy.
Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy.
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抗荧光素 4-4-20 抗原结合片段的分子动力学。
DOI：
10.1021/bi00021a009
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Herron,JN
中科院分区：
文献类型：
--
作者：
Lim,K;Jameson,DM;Gentry,CA;Herron,JN
摘要：采用时间分辨荧光实验研究了一种结合荧光半抗原荧光素的高亲和单克隆抗体（4-4-20）的抗原结合片段（Fab）的动态特性。检测了非配体的Fab和Fab与荧光素的非荧光类似物（荧光胺，FLM）的络合物。荧光极化探针[5-[[2-[（碘乙酰基）氨基]乙基]氨基]萘-1-磺酸（AEDANS）共价连接到Fab的c端。实验在三种不同的温度（10、25和35℃）下进行，并收集了在每种温度下FLM与Fab的五种不同摩尔比的相位调制数据集。然后使用全局分析从这些数据中提取荧光寿命和旋转相关时间的值。在寿命分析中，AEDANS的发射符合洛伦兹寿命分布（r= 15.6 ns，分布宽度=3.4 ns，均为25℃），这表明探针经历了异质环境。各向异性分析表明存在两种不同的旋转分量。第一个是由于Fab的全局运动，在25℃时显示出约33 ns的旋转相关时间（），该成分相对不受抗原结合的影响。第二个旋转分量归因于Fab内的局部或节段运动，并且在25℃下显示出1.1 ns的旋转相关时间(2)，该值在抗原结合后增加了50%以上，这一结果与同一Fab -荧光素系统的分子动力学模拟一致[Lim & Herron (1995) Biochemistry 34, 6962-6974]，此外，统计分析表明，在95%的置信水平下，这种增加是显著的。
Revised Manuscript Received February 28, 1995® abstract: Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4—4—20) which binds the fluorescent hapten fluorescein. Both the unliganded Faband a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization probe [5-[[2-[(iodoacetyl) amino] ethyl] amino] naphthalene-1-sulfonic acid, AEDANS] was covalently attached tothe C-terminus of the Fab. Experiments were performed at three different temperatures (10, 25, and 35 C), and phase-modulation data sets were collected for five different molar ratios of FLM to Fab at each temperature. Global analyses were then used to extract values for fluorescence lifetime and rotational correlation time from these data. In the lifetime analysis the best fit was obtained when the emission of AEDANS was described by a Lorentzian distribution of lifetimes (r= 15.6 ns, distribution width=3.4 ns, both at 25 C), which suggested that the probe experienced a heterogeneous environment. Anisotropy analyses suggested that two different rotational components were present. The first was attributed to the global motion of the Fab and exhibited a rotational correlation time () of ca. 33 ns at 25 C. This component was relatively unaffected by antigen binding. Thesecond rotational component was attributed to the local or segmental motion within the Fab and exhibited a rotational correlation time (2) of 1.1 ns at 25 C. This value increased by more than 50% upon antigen binding, a result which was consistent with molecular dynamics simulations of the same Fab—fluorescein system [Lim & Herron (1995) Biochemistry 34, 6962—6974], Furthermore, statistical analysis showed that this increase was significant at the95% confidence level.