Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR.
Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR.
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通过带选择性同核解偶联 (1) H NMR 测定肽的残基特异性酸解离常数。
DOI：
10.1021/ac070976v
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发表时间：
2007
影响因子：
7.4
通讯作者：
Rabenstein,DallasL
中科院分区：
文献类型：
--
作者：
Wang,Jing;Rabenstein,DallasL
肽中氨基酸残基的侧链酸性基团的酸解离常数可以通过1H NMR测定，前提是可以分辨位于酸性基团附近的碳键报告质子的共振。在这里，我们报告的频带选择性homonuclear-decoupled（BASHD）TOCSY实验的分辨率增加，大大扩展了NMR方法的应用范围，用于测定含有多个残基的相同氨基酸的肽的残基特异性，侧链酸解离常数。化学位移-pH滴定曲线是从BASHD-TOCSY光谱中报告质子的交叉峰获得的，测量结果为pH值的函数。该方法基于使用主链CαH质子共振化学位移的序列依赖性差异，以及BASHD-TOCSY光谱中从CαH多重峰塌陷到F1维单峰的分辨率增加，以解析侧链报告质子的共振。通过测定十六肽Ac-SRGKAKVKAKVKDQTK-NH 2中六个赖氨酸残基的每个侧链铵基团的残基特异性pKa值，证明了该方法的应用。赖氨酸侧链Cε H2报告质子的化学位移-pH滴定曲线是从BASHD-TOCSY光谱中解析的Cα H-C ε H2 TOCSY交叉峰获得的。六个铵基团的相对酸度也确定从残留物特定的化学位移-pH滴定数据的pH值无关的方法，并使用残留物特定的pKa值的质子化微种的分数浓度的计算也进行了描述。
Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift−pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH. The method is based on using sequence-dependent differences in the chemical shifts of resonances for the backbone CαH protons and the increased resolution in BASHD-TOCSY spectra from collapse of CαH multiplets to singlets in theF1 dimension to resolve resonances for the side-chain reporter protons. Application of the method is demonstrated by determination of residue-specific pKAvalues for each of the side-chain ammonium groups of the six lysine residues in the hexadecapeptide Ac-SRGKAKVKAKVKDQTK-NH2. Chemical shift−pH titration curves were obtained for the lysine side-chain CεH2reporter protons from their resolved CαH−CεH2TOCSY cross-peaks in BASHD-TOCSY spectra. Relative acidities of the six ammonium groups were also determined from the residue specific chemical shift−pH titration data by a pH-independent method, and calculation of fractional concentrations of protonation microspecies using the residue-specific pKAs is also described.