Moran Haley M, Manriquez-Sandoval Edgar, Sharma Piyoosh, Fried Stephen D, Gillilan Richard E
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218.
Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218.
PRX Life. 2024 Sep;2(3). doi: 10.1103/prxlife.2.033011. Epub 2024 Sep 9.
One of the planet's more understudied ecosystems is the deep biosphere, where organisms can experience high hydrostatic pressures (30-110 MPa); yet, by current estimates, these subsurface and deep ocean zones host the majority of the Earth's microbial and animal life. The extent to which terrestrially relevant pressures up to 100 MPa deform most globular proteins - and which kinds - has not been established. Here, we report the invention of an experimental apparatus that enables structural proteomic methods to be carried out at high pressures for the first time. The method, called high-pressure limited proteolysis (Hi-P LiP), involves performing pulse proteolysis on whole cell extracts brought to high pressure. The resulting sites of proteolytic susceptibility induced by pressure are subsequently read out by sequencing the peptide fragments with tandem liquid chromatography-mass spectrometry. The method sensitively detects pressure-induced structural changes with residue resolution and on whole proteomes, providing a deep and broad view of the effect of pressure on protein structure. When applied to a piezo-sensitive thermophilic bacterium, , we find that ca. 40% of its soluble proteome is structurally perturbed at 100 MPa. Proteins with lower charge density are more resistant to pressure-induced deformation, as expected; however, contrary to expectations, proteins with lower packing density (i.e., more voids) are also more resistant to deformation. Furthermore, high pressure has previously been shown to preferentially alter conformations around active sites. Here, we show this is also observed in Hi-P LiP, suggesting that the method could provide a generic and unbiased modality to detect binding sites on a proteome scale. Hence, datasets of this kind could prove useful for training emerging AI models to predict cryptic binding sites with greater accuracy.
地球上研究较少的生态系统之一是深层生物圈，在那里生物体可承受高静水压力（30 - 110兆帕）；然而，据目前估计，这些地下和深海区域承载着地球上大部分的微生物和动物生命。高达100兆帕的与陆地相关的压力能使大多数球状蛋白质发生何种程度的变形以及哪些种类的蛋白质会发生变形，目前尚未明确。在此，我们报告了一种实验装置的发明，该装置首次使结构蛋白质组学方法能够在高压下进行。这种方法称为高压有限蛋白酶解（Hi - P LiP），包括对处于高压状态的全细胞提取物进行脉冲蛋白酶解。随后，通过串联液相色谱 - 质谱对肽段进行测序，读出由压力诱导产生的蛋白水解敏感位点。该方法能以残基分辨率灵敏地检测压力诱导的结构变化，并可应用于整个蛋白质组，从而深入广泛地了解压力对蛋白质结构的影响。当应用于一种对压力敏感的嗜热细菌时，我们发现其约40%的可溶性蛋白质组在100兆帕压力下结构受到扰动。正如预期的那样，电荷密度较低的蛋白质对压力诱导的变形更具抗性；然而，与预期相反的是，堆积密度较低（即空隙较多）的蛋白质对变形也更具抗性。此外，此前已表明高压会优先改变活性位点周围的构象。在此，我们表明在Hi - P LiP中也观察到了这一点，这表明该方法可为在蛋白质组规模上检测结合位点提供一种通用且无偏差的方式。因此，这类数据集可能有助于训练新兴的人工智能模型，以更高的准确率预测隐秘的结合位点。
