Thermodynamic description of bacterial metal-sensing: A cellular logic for metals

Abstract

Almost half of all enzymes require a metal ion to function; yet most proteins will bind the ‘wrong’ metal more tightly than the ‘right’ one. Cells can achieve correct metalation provided the tight binding, competitive metals are kept less available than the weak binding ones. However, ‘metal availability’ has been challenging to define and measure. The purpose of this thesis was to address these challenges.
Bacterial metal-sensing DNA-binding transcriptional regulators are tuned to respond within a narrow range of intracellular availabilities of the metals they sense. Therefore, sensors can be used as a window through which to observe intracellular metal availability in a bacterial cell.
Two sensors from the set of metalloregulators in Salmonella, MntR and Fur, have been characterised in vitro to determine their affinities for their cognate metals and for DNA. In order to calculate sensor responses to metals, a thermodynamics-based mathematical model was developed and applied to MntR and Fur, as well as to the other sensors within the set. From the sensitivities of the metal sensors, the standard free energies for metal complex formation in Salmonella cells were determined. Notably, these free energies follow the inverse of the Irving-Williams series: The more competitive the metal, the more favourable the free energy for metal complex formation and hence the lesser availability to which the cognate sensor is attuned.
These results not only illustrate a cellular logic for metalation, but also enable predictions of what metal a molecule of interest will bind in vivo and the fractional metalation state of that molecule. Here, this has been exemplified by examining how cobalt partitions into the vitamin B12 biosynthetic pathway in Salmonella. As metal availability can change in response to growth conditions, a methodology to refine and adapt the model to bespoke culture conditions has also been tested.