Directed evolution of Nickel and Iron binding sites in the synthetic, chimeric NiFe protein
This is the title of the project for which we have secured competitive funding from Britain's Royal Society. According to the Society's literature, "the Royal Society was formed over 350 years ago by a group of people who wanted to share ideas about the natural world by using experiments and observations". Today, the Society is renowned for its prestigious cohort of "Fellows of the Royal Society"; distinguished scientists who have been elected on the basis of the impact their research by leading experts from across the world of Science. Famous Fellows from the past include Sir Isaac Newton, Robert Hooke and Charles Darwin and from their annual announcements every January, last year included Manjul Bhargava for his work on number theory, Veronique Gouverneur, for her work on fluorine chemistry and Jack Szostak, for his work on molecular genetics and in particular DNA replication and evolution. The current The current President of the Royal Society is Dr Venki Ramakrishnan, who received the Nobel Prize for his work on determination of the structure of the ribosome. Partnership grants are awarded to provide school students with an opportunity to explore a research problem alongside a University research group. In this case, students will be mentored by Mr Blackham at the NLA and Dr. Dyer at the UTC. The project is an ongoing research problem that has been investigated by two Masters Level students, James Florence (now a PhD student in Professor Hornby's lab at the University of Sheffield) and Alex Wakeman. The protein called NiFe is encoded by a small gene which has been made by chemical synthesis and combines a Ni binding domain (NBD) from the human transcription factor nonO and the complete sequence of the Fe binding electron transfer protein, rubredoxin. The N terminus of the nonO protein, sometimes called p54nrb, is Glutamine (Gln,Q), Proline (Pro, P) and Histidine (His,H) rich as you can see below. The His residues coordinate the Ni ion, and often this property is utilised by adding six consecutive His residues to the N or C terminus of a recombinant protein, to facilitate its purification on a Ni chromatography resin (the most popular is called Ni-NTA).
MQSNKTFNLEKQNHTPRKHHQHHHQQQHHQQQQQQPPPPPIPANG
The function of the NBD is unclear, but it allows us to explore the ways in which metal ions, which are widely used in Chemistry as catalysts, join forces with proteins (polypeptide chains) to enhance the catalytic and redox properties of proteins early on in the evolution of Life on Earth. A possible 3D model for the complex with Ni can be derived from a related crystal structure (below), discussed in a review of Ni binding proteins found in E.coli. This will form part of the project.
The Fe or iron, binding domain (IBD) is derived from the rubredoxin redox protein from thermophilic microbe, Thermotoga maritima. It has a structure shown below (from a closely related species (PDB Ref to add), and a polypeptide sequence shown below the image, following on from the p54nrb sequence.
These are the synthetic proteins, but what are the questions? Here is a taster, rather than a comprehensive programme of work. Darwinian evolution is based on an intrinsically error-prone process that copies gene sequences from one generation to the next. When a selective advantage arises that promotes the advantage of a particular mutant within a population, that mutant will begin to dominate and eventually replace (in some situations), the parental gene (or genome). We have developed (James's PhD thesis) a mutant form of a Polymerase Chain Reaction enzyme that introduces errors into any gene, allowing us to "evolve" our NiFe protein in the test-tube: thereby compressing evolutionary time from many years into days. Can we use this process of molecular evolution to change NiFe into CoFe for example?
The second strand of the study is to understand the mechanism underlying metal ion catalysis of reactions such as the decomposition of hydrogen peroxide. Ions like manganese (in a number of oxidation states) are able to catalyse the decomposition reaction very efficiently. So why did catalase evolve? Does it make a metal ion better or does it use a different catalytic pathway? What are the chemical and physical parameters that confer catalytic power on metal ions in solution (and in the solid phase)? How does our understanding on the coordination of metal ions in solution help us understand catalysis? And what is the connection between catalysis in chemistry and biochemistry to the quantum mechanical models of atomic orbitals?
I hope you can see we have an opportunity to dig a little deeper into the science behind catalysis in both the test tube and the cell. I will begin to populate the blog site myself and then I will hand over to you!
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