Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University , Frankfurt, 60438, Germany.
J Am Chem Soc. 2013 Sep 18;135(37):13796-803. doi: 10.1021/ja405244v. Epub 2013 Sep 6.
Proteins and nucleic acids maintain the crowded interior of a living cell and can reach concentrations in the order of 200-400 g/L which affects the physicochemical parameters of the environment, such as viscosity and hydrodynamic as well as nonspecific strong repulsive and weak attractive interactions. Dynamics, structure, and activity of macromolecules were demonstrated to be affected by these parameters. However, it remains controversially debated, which of these factors are the dominant cause for the observed alterations in vivo. In this study we investigated the globular folded peptidyl-prolyl isomerase Pin1 in Xenopus laevis oocytes and in native-like crowded oocyte extract by in-cell NMR spectroscopy. We show that active Pin1 is driven into nonspecific weak attractive interactions with intracellular proteins prior to substrate recognition. The substrate recognition site of Pin1 performs specific and nonspecific attractive interactions. Phosphorylation of the WW domain at Ser16 by PKA abrogates both substrate recognition and the nonspecific interactions with the endogenous proteins. Our results validate the hypothesis formulated by McConkey that the majority of globular folded proteins with surface charge properties close to neutral under physiological conditions reside in macromolecular complexes with other sticky proteins due to molecular crowding. In addition, we demonstrate that commonly used synthetic crowding agents like Ficoll 70 are not suitable to mimic the intracellular environment due to their incapability to simulate biologically important weak attractive interactions.
蛋白质和核酸维持着活细胞内部的拥挤环境，其浓度可达 200-400g/L，这会影响环境的物理化学参数，如粘度、流体力学以及非特异性强斥力和弱引力。研究表明，大分子的动力学、结构和活性会受到这些参数的影响。然而，哪种因素是导致体内观察到的改变的主要原因，这在学术界仍存在争议。在这项研究中，我们通过细胞内 NMR 光谱法研究了非洲爪蟾卵母细胞中的球形折叠肽脯氨酰顺反异构酶 Pin1 以及类似天然的拥挤卵母细胞提取物中的 Pin1。我们发现，在底物识别之前，活性 Pin1 会与细胞内的蛋白质发生非特异性的弱引力相互作用。Pin1 的底物识别位点具有特异性和非特异性的引力相互作用。PKA 对 WW 结构域丝氨酸 16 的磷酸化会破坏底物识别以及与内源性蛋白质的非特异性相互作用。我们的结果验证了 McConkey 提出的假设，即在生理条件下表面电荷性质接近中性的大多数球形折叠蛋白由于分子拥挤而与其他粘性蛋白存在于大分子复合物中。此外，我们还证明了常用的合成拥挤剂（如 Ficoll 70）由于无法模拟重要的生物弱引力，因此不适合模拟细胞内环境。
