Understanding mis-metalation of native versus heterologously expressed protein

所有已知的生命依赖于金属、微生物和植物。不同金属的特殊化学性质扩大了蛋白质可以在没有辅助的情况下发挥作用的能力。这需要化学上正确的金属(S)与每种金属蛋白相关联。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会比正确的金属更紧密地结合错误的金属。我们现在认识到，正确的金属化是蛋白质及其生物环境共同作用的结果。蛋白质与其他分子竞争每一种金属。几千年来，这种竞争已经演变成通常正确的金属获胜，错误金属化在很大程度上被避免：但这是否意味着蛋白质在不同生物中表达时容易错误金属化，或者如果细胞环境以某种方式改变？DNA结合的金属传感器被调整为与细胞内环境竞争可用金属(自然化学生物学2017 13：409-417)。在BBSRC的支持下，在近十年的时间里，收集了一系列DNA结合金属传感器的热力学数值(来自沙门氏菌)，从而可以计算每种金属的细胞内竞争幅度(自然化学生物学2019 15：241-0249)。通过参考这些值，就有可能预测金属蛋白的金属化。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(自然通讯2021 12：1195)。现在有必要在其他生物体中测试这样的计算。一些金属被嵌入到余因中。为辅因子提供金属的蛋白质，如钴和维生素B12，也必须以某种方式获得正确的金属。值得注意的是，随着越来越多的人转向低肉类饮食，植物不会使B12产生对这种维生素供应的兴趣。大肠杆菌也不产生B12，但COI已经(通过引入30多个基因)改造了大肠杆菌，产生了一种高产B12的菌株。我们已经证明，在合成的大肠杆菌系统中，B12的钴输送蛋白容易与锌错误金属化，但当细胞补充多余的钴时，错误金属化就会被克服。这种错误的金属化是引入的蛋白质和细胞环境之间不匹配的功能吗？我们将以一种适合于确定传感器的金属亲和力和DNA亲和力的方式，表达、纯化和制备与锌和钴敏感以及B12的钴传递有关的推导或已知的根瘤菌蛋白。传感器的丰度也将被确定。因此，将产生一个混合根瘤菌金属化计算器，它可以确定B12途径与钴和锌(或铁)在这个天然宿主系统中的金属化和错金属化。将测量B12在自由生活的根瘤菌和根瘤中的产量并计算金属化。这项工作的一个中心目的是进一步开发(并举例说明)在生物学中具有广泛应用/影响的金属化计算器的实用性。这项工作对植物性饮食中B12的供应也有影响(参见关于推进更高TRL的结束语)。根瘤菌在豆科植物的根瘤中产生B12，因此钴促进豆类的生长。设计种子敷料(可能含有钴或使用锌螯合剂)以增强固氮的能力也对全球可持续发展具有影响，因为氮肥的制造占能源需求的2%。工业生物技术和生物能源BBSRC网络的第一阶段和现在的直接第二阶段，其明确目的是加速利用生物金属方面的这种进展来支持生物技术。

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.