Understanding mis-metalation of native versus heterologously expressed protein

所有已知的生命都依赖于金属、微生物和植物。不同金属的特殊化学性质扩大了蛋白质独立完成任务的范围。这需要化学上正确的金属与每种金属蛋白相结合。人们很容易认为这种金属化是蛋白质本身的唯一功能，但纯化的蛋白质通常比正确的蛋白质结合错误的金属要紧密许多个数量级。我们现在认识到，正确的金属化作用是蛋白质及其生物环境的综合作用。蛋白质与其他分子争夺每种金属。几千年来，这种竞争已经进化到正确的金属通常会获胜，而错误的金属化在很大程度上是避免的：但这是否意味着蛋白质在不同的生物体中表达时，或者如果细胞环境发生了某种改变，就容易发生错误的金属化呢？dna结合金属传感器被调整为与细胞内环境竞争可用金属（Nature Chemical Biology 2017 13:409-417）。在近十年的时间里，在BBSRC的支持下，为一组dna结合金属传感器（来自沙门氏菌）收集了一系列热力学值，从而可以计算每种金属的细胞内竞争程度（Nature Chemical Biology 2019:241-0249）。根据这些值，可以对金属蛋白的金属化进行预测。我们最近开发了一种金属化计算器，可以在沙门氏菌和密切相关的大肠杆菌中提供这种计算（Nature Communications 2021 12: 1195）。现在有必要在其他生物体中检验这种计算。有些金属嵌入辅因子中。为辅因子提供金属的蛋白质，如为维生素B12提供钴的蛋白质，也必须以某种方式获得正确的金属。值得注意的是，植物不会产生B12，对这种维生素的供应产生兴趣，因为越来越多的人转向低肉饮食。大肠杆菌也不产生B12，但CoI已经改造（通过引入30多个基因）大肠杆菌，使其产生了一种B12高产菌株。我们已经证明，在合成大肠杆菌系统中，B12的钴递送蛋白容易与锌发生错金属化，但当细胞补充了多余的钴时，错金属化就会被克服。这种错金属化是引入的蛋白质与细胞环境不匹配的结果吗？我们将表达、纯化和制备推断的或已知的根瘤菌蛋白，这些蛋白参与锌和钴的传感，以及钴对B12的递送，以适合于确定传感器的金属亲和力和dna亲和力的方式。传感器的丰度也将被确定。因此，一个杂交根瘤菌金属化计算器将产生，可以确定B12途径与钴和锌（或铁）的金属化和错金属化。将测量游离根瘤菌和根瘤中B12的产量并计算金属化。这项工作的一个中心目的是进一步发展（并举例说明）金属化计算器在生物学中的广泛应用/含义。这项工作也对那些以植物性饮食为基础的人的B12供应有影响（参见关于提高trl的结束语）。根瘤菌在豆科植物根瘤中产生B12，因此钴促进豆科植物生长。设计种子敷料（可能含有钴或锌螯合剂）以增强固氮的能力也对全球可持续性产生影响，因为氮肥的制造占能源需求的2%。PI和CoI指导了BBSRC工业生物技术和生物能源网络的第一阶段，现在正在指导第二阶段，其明确目的是加速利用生物金属领域的这些进步来支持生物技术。

All known life depends on metals, microbial and plant. The special chemical properties of different metals expand the repertoire of what proteins can do unaided. This requires the chemically correct metal(s) to associate with each metalloprotein. It is tempting to assume that such metalation is a sole function of the proteins themselves, but purified proteins typically bind wrong metals many orders of magnitude more tightly than the correct ones. We now appreciate that correct metalation is a combined function of proteins and their biological surroundings. Proteins compete with other molecules for each metal. Over the millennia this competition has evolved such that the correct metals generally win and mis-metalation is largely avoided: But does this mean that proteins are liable to mis-metalation when expressed in different organisms or if the cellular milieu is somehow altered? DNA-binding metal sensors are tuned to compete for available metal with the intracellular milieu (Nature Chemical Biology 2017 13:409-417). Over the course of nearly a decade, supported by the BBSRC, a series of thermodynamic values were collected for a set of DNA-binding metal sensors (from Salmonella), making it possible to calculate the magnitude of intracellular competition for each metal (Nature Chemical Biology 2019 15:241-0249). By reference to these values, it has become possible to make predictions about the metalation of metalloproteins. We have recently developed a metalation calculator that provides such calculations in Salmonella and closely related E. coli (Nature Communications 2021 12: 1195). It is now necessary to test such calculations in other organisms.Some metals are embedded into cofactors. Proteins that supply metals to cofactors, such as cobalt to vitamin B12, must also somehow acquire the correct metal. Notably, plants do not make B12 generating interest in the supply of this vitamin for the increasing numbers of individuals turning to low meat diets. E. coli also does not make B12 but the CoI has engineered (by introduction of more than 30 genes) E. coli to generate a B12 hyper-producing strain. We have shown that in the synthetic E. coli system the cobalt delivery protein for B12 is susceptible to mis-metalation with zinc but mismetalation is overcome when cells are supplemented with surplus cobalt. Is such mis-metalation a function of a mis-match between the introduced proteins and the cellular milieu? We will express, purify and prepare the deduced or known Rhizobium proteins involved in sensing zinc and cobalt, and in cobalt delivery for B12, in a manner suitable for determining metal affinities and DNA-affinities for the sensors. Sensor abundance will also be determined. A hybrid Rhizobium metalation calculator will thus be produced that can determine metalation and mis-metalation of the B12 pathway with cobalt and zinc (or iron) in this native host system. B12 production will be measured and metalation calculated both in free living Rhizobium and in root nodules. One central purpose of this work is to further develop (and exemplify the utility of) the metalation calculator with widespread applications/implications in biology. This work also has implications for the supply of B12 to those on plant-based diets (refer to closing statement on advancing to higher TRLs). Rhizobium makes B12 in nodules of leguminous plant roots and thus cobalt promotes legume growth. An ability to design seed dressings (potentially containing cobalt or with a zinc chelator), to enhance nitrogen fixation also has implications for global sustainability since the manufacture of nitrogen fertiliser represents up to 2% of energy demands. The PI and CoI directed phase I, and now direct phase II, of a BBSRC Network in Industrial Biotechnology and Bioenergy with the express purpose of accelerating the exploitation of such advances in metals in biology to support biotechnology.