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 使用抗肽抗体作为蛋白质折叠的探针和 动力学。有必要首先研究其发病机制。 完整蛋白和抗肽抗体之间的交叉反应。它是 也有必要开发一种方便的方法来研究结构 当蛋白质与抗肽抗体结合时。我们会研究 牛胰腺胰蛋白酶抑制物(BPTI)，一种已经被 以生物物理和结构方法为特征的。 将使用差分圆二色谱(CD)来研究 低分辨率下的交叉反应机理。动力学和 交叉反应的热力学将被描述。我们还将 确定多肽的能力之间是否存在相关性 折叠，通过CD和核磁共振(核磁共振)判断，并能够 以引发交叉反应抗体。 酰胺质子交换，结合二维核磁共振波谱 (2D-核磁共振)将用于研究BPTI的结构，而它势必 抗肽抗体。BPTI抗体中的氢/氢交换 将允许发生复杂的情况。然后，BPTI将与 抗体，2D-核磁共振将被用来定量交换的程度 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.