Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Am Chem Soc. 2010 Dec 15;132(49):17411-25. doi: 10.1021/ja9067035. Epub 2010 Nov 19.
This paper combines two techniques--mass spectrometry and protein charge ladders--to examine the relationship between the surface charge and hydrophobicity of a representative globular protein (bovine carbonic anhydrase II; BCA II) and its rate of amide hydrogen-deuterium (H/D) exchange. Mass spectrometric analysis indicated that the sequential acetylation of surface lysine-ε-NH3(+) groups--a type of modification that increases the net negative charge and hydrophobicity of the surface of BCA II without affecting its secondary or tertiary structure--resulted in a linear decrease in the aggregate rate of amide H/D exchange at pD 7.4, 15 °C. According to analysis with MS, the acetylation of each additional lysine generated between 1.4 and 0.9 additional hydrogens that are protected from H/D exchange during the 2 h exchange experiment at 15 °C, pD 7.4. NMR spectroscopy demonstrated that none of the hydrogen atoms which became protected upon acetylation were located on the side chain of the acetylated lysine residues (i.e., lys-ε-NHCOCH3) but were instead located on amide NHCO moieties in the backbone. The decrease in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs of the charge ladder were the least thermostable (as measured by differential scanning calorimetry). This observation--that faster rates of exchange are associated with slower rates of denaturation--is contrary to the usual assumptions in protein chemistry. The fact that the rates of H/D exchange were similar for perbutyrated BCA II (e.g., [lys-ε-NHCO(CH2)2CH3]18) and peracetylated BCA II (e.g., [lys-ε-NHCOCH3]18) suggests that the electrostatic charge is more important than the hydrophobicity of surface groups in determining the rate of H/D exchange. These electrostatic effects on the kinetics of H/D exchange could complicate (or aid) the interpretation of experiments in which H/D exchange methods are used to probe the structural effects of non-isoelectric perturbations to proteins (i.e., phosphorylation, acetylation, or the binding of the protein to an oligonucleotide or to another charged ligand or protein).
本文结合两种技术——质谱和蛋白质电荷梯——研究了代表性球状蛋白（牛碳酸酐酶 II；BCA II）表面电荷和疏水性与其酰胺氢-氘（H/D）交换速率之间的关系。质谱分析表明，表面赖氨酸-ε-NH3(+)基团的顺序乙酰化——一种增加表面负电荷和疏水性而不影响其二级或三级结构的修饰类型——导致在 pD 7.4、15°C 时酰胺 H/D 交换的总速率呈线性下降。根据 MS 分析，在 15°C、pD 7.4 的 2 小时交换实验中，每个额外赖氨酸的乙酰化产生了 1.4 到 0.9 个额外的氢原子，这些氢原子在交换实验中受到保护而不发生 H/D 交换。NMR 光谱证明，在乙酰化后受到保护的氢原子并不位于乙酰化赖氨酸残基的侧链上（即 lys-ε-NHCOCH3），而是位于骨架中的酰胺 NHCO 部分。与乙酰化相关的交换速率下降与热稳定性下降平行：电荷梯中交换速率最慢的梯级热稳定性最低（通过差示扫描量热法测量）。这种观察结果——即较快的交换速率与较慢的变性速率相关——与蛋白质化学中的常见假设相反。事实上，H/D 交换的速率对于过丁酸化 BCA II（例如，[lys-ε-NHCO(CH2)2CH3]18）和过乙酰化 BCA II（例如，[lys-ε-NHCOCH3]18）相似，这表明静电电荷比表面基团的疏水性更重要在确定 H/D 交换速率方面。这些静电效应对 H/D 交换动力学的影响可能会使（或有助于）解释实验复杂化，其中 H/D 交换方法用于探测非等电扰动对蛋白质的结构影响（即磷酸化、乙酰化或蛋白质与寡核苷酸或另一种带电荷的配体或蛋白质的结合）。
