NMR PROTON EXCHANGE STUDIES OF ANTIBODY CROSS-REACTIONS

这项拟议工作的长期目标是建立一个框架， 用于使用抗肽抗体作为蛋白质折叠的探针， 动力学 首先有必要研究 完整蛋白质和抗肽抗体之间的交叉反应。 是 也有必要发展一种方便的方法来研究结构 蛋白质与抗肽抗体结合的时候。 我们将研究 牛胰蛋白酶抑制剂（BPTI），一种蛋白质， 通过生物物理学和结构学方法进行了充分表征。 差示圆二色性（CD）光谱将用于研究 低分辨率下交叉反应的机理。 的动力学和 将表征交叉反应的热力学。 我们还将 确定肽的能力之间是否存在相关性， 倍，如通过CD和核磁共振（NMR）判断的， 引发交叉反应的抗体。 酰胺质子交换，结合二维NMR光谱 （2D-NMR）将用于研究BPTI的结构，而它与 抗肽抗体。 BPTI-抗体中的氢/氘交换 将允许发生复杂。 然后，BPTI将与 抗体，2D-NMR将用于定量交换的程度， BPTI中的单个酰胺质子。

The long-term objective of this proposed work is to establish a framework for using anti-peptide antibodies as probes of protein folding and dynamics. It is necessary first to investigate the mechanism of cross-reaction between intact proteins and anti-peptide antibodies. It is also necessary to develop a convenient method for studying the structures of proteins while they are bound to anti-peptide antibodies. We will study bovine pancreatic trypsin inhibitor (BPTI), a protein that has been well-characterized by biophysical and structural methods. Difference circular dichroism (CD) spectroscopy will be used to investigate the mechanism of cross-reaction at low resolution. The kinetics and thermodynamics of cross-reaction will be characterized. We will also determine if there is a correlation between the ability of peptides to fold, as judged by CD and nuclear magnetic resonance (NMR), and the ability of peptides to elicit cross-reacting antibodies. Amide proton exchange, combined with two-dimensional NMR spectroscopy (2D-NMR) will be used to study the structure of BPTI, while it is bound to anti-peptide antibodies. Hydrogen/deuterium exchange in the BPTI-antibody complex will be allowed to occur. Then, BPTI will be separated from antibody, and 2D-NMR will be used to quantitate the extent of exchange for individual amide protons in BPTI.