Department of Chemistry, University of California, Riverside, California 92521, United States.
Biochemistry. 2012 Feb 28;51(8):1796-802. doi: 10.1021/bi2018199. Epub 2012 Feb 10.
A simple mass spectrometry-based method capable of examining protein structure called SNAPP (selective noncovalent adduct protein probing) is used to evaluate the structural consequences of point mutations in naturally occurring sequence variants from different species. SNAPP monitors changes in the attachment of noncovalent adducts to proteins as a function of structural state. Mutations that lead to perturbations to the electrostatic surface structure of a protein affect noncovalent attachment and are easily observed with SNAPP. Mutations that do not alter the tertiary structure or electrostatic surface structure yield similar results by SNAPP. For example, bovine, porcine, and human insulin all have very similar backbone structures and no basic or acidic residue mutations, and the SNAPP distributions for all three proteins are very similar. In contrast, four variants of cytochrome c (cytc) have varying degrees of sequence homology, which are reflected in the observed SNAPP distributions. Bovine and pigeon cytc have several basic or acidic residue substitutions relative to horse cytc, but the SNAPP distributions for all three proteins are similar. This suggests that these mutations do not significantly influence the protein surface structure. On the other hand, yeast cytc has the least sequence homology and exhibits a unique, though related, SNAPP distribution. Even greater differences are observed for lysozyme. Hen and human lysozyme have identical tertiary structures but significant variations in the locations of numerous basic and acidic residues. The SNAPP distributions are quite distinct for the two forms of lysozyme, suggesting significant differences in the surface structures. In summary, SNAPP experiments are relatively easy to perform, require minimal sample consumption, and provide a facile route for comparison of protein surface structure between highly homologous proteins.
一种简单的基于质谱的方法，可以检测蛋白质结构，称为 SNAPP（选择性非共价加合物蛋白探测），用于评估来自不同物种的天然存在序列变异体中的点突变对蛋白质结构的影响。SNAPP 监测非共价加合物与蛋白质结合的变化作为结构状态的函数。导致蛋白质静电表面结构扰动的突变会影响非共价结合，并且很容易通过 SNAPP 观察到。不会改变三级结构或静电表面结构的突变通过 SNAPP 产生相似的结果。例如，牛胰岛素、猪胰岛素和人胰岛素都具有非常相似的骨架结构，没有碱性或酸性残基突变，并且这三种蛋白质的 SNAPP 分布非常相似。相比之下，细胞色素 c（cytc）的四个变体具有不同程度的序列同源性，这反映在观察到的 SNAPP 分布中。牛和鸽子 cytc 相对于马 cytc 有几个碱性或酸性残基取代，但这三种蛋白质的 SNAPP 分布相似。这表明这些突变不会显著影响蛋白质表面结构。另一方面，酵母 cytc 的序列同源性最低，表现出独特的 SNAPP 分布，尽管相关。溶菌酶观察到的差异更大。鸡和人溶菌酶具有相同的三级结构，但许多碱性和酸性残基的位置存在显著差异。两种形式的溶菌酶的 SNAPP 分布截然不同，表明表面结构存在显著差异。总之，SNAPP 实验相对容易进行，需要的样品消耗最少，并为高度同源蛋白质之间的蛋白质表面结构比较提供了简便的途径。
