The Contribution of Protein Dynamics to Antibody Affinity Maturation

免疫系统如何进化出针对几乎任何外来分子或抗原的抗体(Ab)， (AG)，不结合任何自身分子是我们理解免疫学和自身免疫的核心。 我们的基本假设是，亲和力的成熟有助于分子的这一非凡壮举 利用诱导匹配机制进化识别不同靶标的多特异性抗体识别 使用锁和钥匙机制仅识别其目标的特定抗体。这表明 生殖系抗体必须灵活且构象异质性，而成熟的同种抗体必须是 更坚硬，构象更均匀。因此，直接测试假设需要 作为亲和力成熟的函数的蛋白质灵活性和异质性的表征。这已经是 在过去具有挑战性，但现代实验和计算方法最近做到了 可行。事实上，利用三脉冲光子回波峰位移和动态斯托克斯移位光谱，我们已经 结果表明，抗荧光素抗体Ab4-4-20的亲和力成熟显著增强了抗体-Ag 并极大地降低了其构象的异质性。 基于这些结果，我们建议使用生物物理技术(核磁共振、激光)的组合 光谱学，X射线结晶学，等温滴定量热法，表面等离子体共振，和 分子动力学模拟)，以严格测试我们对Ab4-4-20的基本假设。我们也 建议用其他抗荧光素抗体来检验我们假设的普遍性，这些抗体可以结合另一个小分子- 分子模型抗原，更重要的是，还有抗HIV抗体。该提案的具体目标是； 目的1：确定4-4-20是否是从一株能与荧光素结合的柔性种系抗体进化而来的。 Fit机制与刚性的成熟抗体相结合，通过锁和钥匙机制结合荧光素。 目标2：用其他抗荧光素抗体和其他抗荧光素抗体测试我们基本假设的一般性 进化为与模型抗原8-甲氧基芘-1，3，6-三磺酸结合。 目的3：用抗HIV抗体0.5？检验我们的基本假设的一般性。 目的4：建立抗体4-4-20和0.5？进化的综合模型。 这些研究将首次对所有与生物相关的蛋白质进行完整的表征。 时间尺度，测试我们的基本假设，它的普遍性和生理相关性，并揭示 免疫系统如何为生物功能量身定制抗体的分子细节。

How the immune system evolves antibodies (Ab) specific for virtually any foreign molecule or antigen, (Ag), without binding any self-molecules is central to our understanding of immunology and autoimmunity. Our fundamental hypothesis is that affinity maturation contributes to this remarkable feat of molecular recognition by evolving polyspecific Ab that recognize different targets with an induced-fit mechanism into specific antibodies that recognize only their target with a lock-and-key mechanism. This suggests that germline Ab must be flexible and conformationally heterogeneous while their mature counterparts must be more rigid and conformationally homogeneous. Thus, directly testing the hypothesis requires the characterization of protein flexibility and heterogeneity as a function of affinity maturation. This has been challenging in the past, but modern experimental and computational methodologies have recently made it feasible. In fact, using 3-pulse photon echo peak shift and dynamic Stokes shift spectroscopy, we have already demonstrated that affinity maturation of anti-fluorescein Ab, Ab 4-4-20, significantly rigidified the Ab-Ag complex and dramatically reduced its conformational heterogeneity. Based on these results, we propose to use a combination of biophysical techniques (NMR, laser spectroscopy, X-ray crystallography, isothermal titration calorimetry, surface plasmon resonance, and molecular dynamics simulations) to rigorously test our fundamental hypothesis for Ab 4-4-20. We also propose to test the generality of our hypothesis with other anti-fluorescein Abs, Abs that bind another small- molecule model antigen, and importantly, and anti-HIV Ab. The Specific Aims of the proposal are; Aim 1: Determine whether 4-4-20 evolved from a flexible germline Ab that binds fluorescein via an induced- fit mechanism into a rigid mature Ab that binds fluorescein via a lock-and-key mechanism. Aim 2: Test the generality of our fundamental hypothesis with other anti-fluorescein Abs and with Abs that evolved to bind the model antigen 8-methoxypyrene-1,3,6,trisulfonic acid. Aim 3: Test the generality of our fundamental hypothesis with an anti-HIV Ab, 0.5¿. Aim 4: Generate comprehensive models of Ab 4-4-20 and 0.5¿ evolution. These studies will provide the first complete characterization of any protein over all biologically relevant timescales, test our fundamental hypothesis, its generality and its physiological relevance, and reveal the molecular details of how the immune system tailors Abs for biological function.