Directional copper trafficking: In vitro and in vivo studies of metal binding and transfer

A protein is a biological polymer with a complex three-dimensional structure. Many proteins bind metal ions which are usually essential for both structure and function even though the metals constitute typically less than 0.5 % of the total mass of the molecule (approximately one third of all proteins contain metals and these molecules are called metalloproteins). Metal ions have to be incorporated into the correct proteins within cells, and systems (networks of proteins) are emerging via which this is achieved. These regulatory networks of proteins also prevent the potentially damaging effects of the uncontrolled reactivity of metal ions and when these systems are impaired various diseases can result. The proposed studies aim to investigate the proteins which are responsible for ensuring that copper is supplied to the correct destination in a relatively simple model organism. Metal affinities of all of the proteins involved will be determined as will their ability to transfer copper. The effects of designed alterations to the protein structure on metal binding and transfer, protein interactions as well as the influence on physiological activity in the model organism will all be assessed. A detailed appreciation of the factors regulating this important biological process will be obtained.

Homeostatic proteins ensure that metal ions reach the correct location within cells. In doing this they prevent binding at inappropriate sites and any adverse reactivity of the free metal ion. Redox-active metals such as copper can give rise to extremely damaging radicals and therefore regulation in these cases is particularly important. The proposed investigations are aimed at elucidating the molecular features which regulate copper import to the thylakoid of a cyanobacterium; arguably the ideal model system for such studies. Many of the copper binding sites involved in this pathway are similar and thus the factors driving metal transfer in the desired direction are not clear. Whether copper trafficking to its final location is controlled by relative binding constants and protein abundances (simple thermodynamics) or is substantially regulated and activated by protein interactions will be investigated. In order to realise the objectives complementary in vitro and in vivo studies will be performed. The determination of the metal binding constants of all of the proteins involved in copper trafficking, and the effect of site-directed mutations around the active sites, along with the quantification of the proteins in the cell, will provide vital information about thermodynamic factors controlling copper import. Metal transfer between wild type and mutated proteins, analysed in vitro, along with the influence of the same mutations on in vivo interactions assessed using a bacterial two-hybrid assay, will demonstrate the nature and extent of the involvement of intermolecular protein contacts in regulating copper import. The ability to also analyse the effect of these mutations in the cyanobacterium provides an added dimension to the studies which will advance understanding of the factors controlling copper homeostasis in a cell. Crystallographic studies will be used to assess the structural reasons underlying the effects observed with mutated proteins.