Liu Zhenjiu, Cheng Shijun, Gallie Daniel R, Julian Ryan R
Department of Chemistry, University of California, Riverside, California 92521, USA.
Anal Chem. 2008 May 15;80(10):3846-52. doi: 10.1021/ac800176u. Epub 2008 Apr 12.
Mass spectrometry (MS) is emerging as an additional tool for examining protein structure by way of experiments where structurally related mass changes induced in solution are subsequently detected in the gas phase. Selective noncovalent adduct protein probing (SNAPP) is a recent addition to this type of experiment. SNAPP utilizes noncovalent recognition of lysine residues with 18-crown-6 (18C6) to monitor changes in protein structure. It has been observed that the number of 18C6 adducts that attach to a protein is a function of the structure of the protein. The present work seeks to examine the underlying chemistry which controls the differential attachment of 18C6 to lysine by using ubiquitin as a model system. Ubiquitin is a small protein with a structure that has been well characterized by multiple techniques. Site-directed mutagenesis was used to create a series of ubiquitin mutants where the lysine residues were exchanged for asparagine one at a time. These mutants were then evaluated by SNAPP-MS to determine the relative contribution of each lysine as a binding site for 18C6. It was found that attachment of 18C6 is largely controlled by the strength of intramolecular interactions involving lysine residues. Salt bridges provide the greatest interference, followed by hydrogen bonds. In addition to determining the mechanism for SNAPP, insights are provided about the structure of ubiquitin including confirmation of the existence of two dynamic states for the native structure. These results are discussed in relation to the biological functions of ubiquitin.
质谱分析法（MS）正作为一种额外的工具崭露头角，通过实验来研究蛋白质结构，这些实验是在溶液中诱导产生与结构相关的质量变化，随后在气相中进行检测。选择性非共价加合物蛋白质探测法（SNAPP）是这类实验中的最新方法。SNAPP利用18-冠-6（18C6）对赖氨酸残基的非共价识别来监测蛋白质结构的变化。据观察，附着在蛋白质上的18C6加合物的数量是蛋白质结构的函数。目前的工作旨在通过使用泛素作为模型系统来研究控制18C6与赖氨酸差异附着的潜在化学机制。泛素是一种小蛋白质，其结构已通过多种技术得到充分表征。使用定点诱变创建了一系列泛素突变体，其中赖氨酸残基一次一个地被天冬酰胺取代。然后通过SNAPP-MS对这些突变体进行评估，以确定每个赖氨酸作为18C6结合位点的相对贡献。研究发现，18C6的附着在很大程度上受涉及赖氨酸残基的分子内相互作用强度的控制。盐桥产生的干扰最大，其次是氢键。除了确定SNAPP的机制外，还提供了有关泛素结构的见解，包括确认天然结构存在两种动态状态。结合泛素的生物学功能对这些结果进行了讨论。
