T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, United States.
The Johns Hopkins University Biomolecular NMR Center, Johns Hopkins University, Baltimore, Maryland 21218, United States.
J Am Chem Soc. 2020 Apr 1;142(13):6227-6235. doi: 10.1021/jacs.0c00290. Epub 2020 Mar 17.
A hallmark feature of biological lipid bilayer structure is a depth-dependent polarity gradient largely resulting from the change in water concentration over the angstrom length scale. This gradient is particularly steep as it crosses the membrane interfacial regions where the water concentration drops at least a million-fold along the direction of the bilayer normal. Although local water content is often assumed to be a major determinant of membrane protein stability, the effect of the water-induced polarity gradient upon backbone hydrogen bond strength has not been systematically investigated. We addressed this question by measuring the free energy change for a number of backbone hydrogen bonds in the transmembrane protein OmpW. These values were obtained at 33 backbone amides from hydrogen/deuterium fractionation factors by nuclear magnetic resonance spectroscopy. We surprisingly found that OmpW backbone hydrogen bond energies do not vary over a wide range of water concentrations that are characteristic of the solvation environment in the bilayer interfacial region. We validated the interpretation of our results by determining the hydrodynamic and solvation properties of our OmpW-micelle complex using analytical ultracentrifugation and molecular dynamics simulations. The magnitudes of the backbone hydrogen bond free energy changes in our study are comparable to those observed in water-soluble proteins, the H-segment of the leader peptidase helix used in the von Heijne and White biological scale experiments, and several interfacial peptides. Our results agree with those reported for the transmembrane α-helical portion of the amyloid precursor protein after the latter values were adjusted for kinetic isotope effects. Overall, our work suggests that backbone hydrogen bonds provide modest thermodynamic stability to membrane protein structures and that many amides are unaffected by dehydration within the bilayer.
生物脂质双层结构的一个显著特征是深度依赖的极性梯度，主要是由于水浓度在埃尺度上的变化。这个梯度特别陡峭，因为它穿过膜界面区域，在那里水浓度沿着双层法向方向下降至少一百万倍。尽管局部含水量通常被认为是膜蛋白稳定性的主要决定因素，但水诱导的极性梯度对骨架氢键强度的影响尚未得到系统研究。我们通过测量跨膜蛋白 OmpW 中的一些骨架氢键的自由能变化来解决这个问题。这些值是通过核磁共振波谱法从氢/氘分馏因子获得的 33 个骨架酰胺得到的。我们惊讶地发现，OmpW 骨架氢键的能量在一个广泛的水浓度范围内没有变化，这些水浓度是双层界面区域溶剂化环境的特征。我们通过使用分析超速离心和分子动力学模拟来确定我们的 OmpW-胶束复合物的流体力学和溶剂化性质，验证了我们结果的解释。我们研究中骨架氢键自由能变化的幅度与在水溶性蛋白质、von Heijne 和 White 生物尺度实验中使用的先导肽酶螺旋的 H 段以及几个界面肽中观察到的那些相当。我们的结果与在对跨膜α螺旋部分的淀粉样前体蛋白的报告结果一致，后者的值根据动力学同位素效应进行了调整。总的来说，我们的工作表明，骨架氢键为膜蛋白结构提供了适度的热力学稳定性，并且许多酰胺在双层内不受脱水的影响。
