Department of Chemistry, Texas Materials Institute, and Institute for Cell and Molecular Biology , The University of Texas at Austin , 105E 24th Street , STOP A5300, Austin , Texas 78712-1224 , United States.
J Phys Chem B. 2018 Jul 5;122(26):6733-6743. doi: 10.1021/acs.jpcb.8b03907. Epub 2018 Jun 25.
Vibrational spectroscopy is a powerful tool for characterizing the complex noncovalent interactions that arise in biological systems. The nitrile stretching frequency has proven to be a particularly convenient biological probe, but the interpretation of nitrile spectroscopy is complicated by its sensitivity to local hydrogen bonding interactions. This often inhibits the straightforward interpretation of nitrile spectra by requiring knowledge of the molecular-level details of the local environment surrounding the probe. While the effect of hydrogen bonds on nitrile frequencies has been well-documented for small molecules in solution, there have been relatively few studies of these effects in a complex protein system. To address this, we introduced a nitrile probe at nine locations throughout green fluorescent protein (GFP) and compared the mean vibrational frequency of each probe to the specific hydrogen bonding geometries observed in molecular dynamics (MD) simulations. We show that a continuum of hydrogen bonding angles is found depending on the particular location of each nitrile, and that the differences in these angles account for the differences in the measured nitrile frequency. We further observed that the temperature dependence of the nitrile frequencies (measured as a frequency-temperature line slope, FTLS) was a good indicator of the hydrogen bonding interactions of the probe, even in scenarios where the nitrile was involved in complex and restricted hydrogen bonds, both from protein and from water. While constant offsets to the nitrile frequency to all hydrogen bonding environments have been applied before to interpret shifts in nitrile frequency, we show that this is insufficient in systems where the hydrogen bonds may be restricted by the surrounding medium. However, the strength of the observed correlation between nitrile frequency and hydrogen bonding angle suggests that it may be possible to disentangle electrostatic effects and effects of the orientation of hydrogen bonding on the nitrile stretching frequency. Meanwhile, the experimental measurement of the FTLS of the nitrile is an excellent tool to identify changes in the hydrogen bonding interactions for a particular probe.
振动光谱是一种用于描述生物系统中复杂非共价相互作用的强大工具。腈基伸缩频率已被证明是一种特别方便的生物探针，但由于其对局部氢键相互作用的敏感性，腈基光谱的解释变得复杂。这通常会抑制通过要求了解探针周围局部环境的分子水平细节来直接解释腈基光谱。虽然氢键对小分子在溶液中的腈基频率的影响已有很好的记录，但在复杂蛋白质系统中对这些影响的研究相对较少。为了解决这个问题，我们在绿色荧光蛋白 (GFP) 中引入了九个位置的腈基探针，并将每个探针的平均振动频率与分子动力学 (MD) 模拟中观察到的特定氢键几何形状进行了比较。我们表明，取决于每个腈基的特定位置，发现了氢键角度的连续体，并且这些角度的差异解释了测量的腈基频率的差异。我们还观察到，腈基频率的温度依赖性（以频率-温度线斜率 (FTLS) 测量）是探针氢键相互作用的良好指标，即使在腈基参与复杂和受限氢键的情况下也是如此，这些氢键来自蛋白质和水。虽然以前已经应用过将腈基频率的恒定偏移应用于解释腈基频率的偏移，但我们表明，在氢键可能受到周围介质限制的系统中，这是不够的。然而，观察到的腈基频率与氢键角度之间的相关性很强，这表明可能有可能将静电效应和氢键对腈基伸缩频率的取向的影响分开。同时，腈基的 FTLS 的实验测量是识别特定探针的氢键相互作用变化的极好工具。
