Dr D. Dafydd Jones - PhD
The Jones group focus on understanding the molecular basis of protein plasticity in terms of structure, function and folding, and its application to the construction of new protein components and systems. The ultimate aim is to address one of the fundamental questions in biology: how amino acid sequence encodes the information for a protein to fold to its functional 3D structure. Our group collaborates chemists, structural biologists, computer modellers and physicists. Constructing new protein components means our work is closely aligned with the areas of synthetic biology and nanoscience/nanotechnology.
Protein structure, function and folding
The structure, function and folding of a range of proteins are investigated using biochemical, biophysical, structural biology and molecular dynamics. The group focuses on serine proteases, notably the subtilisin class, fluorescent proteins, luciferase and lipoyl domains. Our achievements the first crystal structure of an intracellular subtilisin that revealed an original combined mechanism for protease regulation inside the cell; high resolution structure of one of the most important variants of GFP (EGFP) that reveals alternate conformations of critical functional residues; structural basis of novel protein misfolding events.
Structure of a misfolded dimeric lipoyl domain (left), model of an artificial protein switching scaffold (middle), mechanism of inbuilt inhibition of intracellular subtilisin protease
Construction of artificial protein scaffolds
Structure of an engineered didomain high efficiency energy transfer protein scaffold
The ability to design new proteins with activities not part of the natural repertoire is essential as part of the development of synthetic biology and bionanotechnology. As a general approach for creating tailored protein components with unique but useful properties, we couple the structures and hence functions of normally unrelated proteins so as to link the function. To achieve this, existing proteins will require radical redesign or even the creation of new scaffolds. However, natural proteins provide the inspiration and guidance during construction. Both rational protein engineering and a recently developed transposon-based non-homologous directed evolution method are used to construct artificial protein scaffolds. We use various metal/porphyrin binding proteins (e.g. cyt b 562), DNA binding proteins (e.g. leucine zippers), enzymes (e.g. TEM-1) and fluorescent proteins as the templates for our novel protein switches.
Single molecule studies of electron transfer proteins
Single molecule of electron transfer protein cytochrome b562 engineered to bind between two gold electrode surfaces.
Electron transfer plays a vital role in biology being pivotal to processes such as photosynthesis, respiration and enzyme catalysis. Such proteins essentially work at the single molecule but most approaches at analyse at the bulk level so all the important detail becomes averaged out. Furthermore, given that proteins self assemble, organise and modulate electron transfer system at the single molecule, there is potential for the adaptation for use as nanodevices, including molecular transistors. The Jones groups has been involved in a interdisciplinary research collaboration aimed at investigating the single molecule behaviour of electron transfer proteins. Electron transfer (ET) proteins are engineered for defined and controlled interactions with metal surfaces. This has led to several important developments in the area of molecular electronics and single protein molecule studies. Notably, we have demonstrated for the first time direct coupling of a protein to both electrodes allowing ET to be monitored at the single molecule level. Our studies revealed that cytochrome b562 was remarkably conductive and exhibited transistor-like behaviour with current modulated electrochemically.
Engineering proteins using an expanded genetic code
We are exploring the an expanded of the genetic code to engineer proteins that sample new chemical diversity. The shared genetic code restricts most organisms to the incorporation of the same 20 amino acids into proteins, thus limiting the chemical functionality available to proteins. Expansion of the genetic code to allow the incorporation of potentially useful non-natural amino acids into a growing polypeptide chain in vivo will generate proteins with novel and enhanced physicochemical and biological properties not accessible in Nature. This in turn will provide new approaches for studying both the molecular and cellular aspect of protein function and for adapting proteins for biotechnological applications.
Transposon-based methods for directed evolution
We have also developed a range of transposon-based technologies to sample various different mutational events not normally sampled during directed evolution. The method can be used to insert or delete multiples of three nucleotides resulting in the in-frame deletion or insertion of amino acids at random positions in a protein. Indel mutations alter the structure and hence function of protein in ways not accessible to substitution mutations alone thus expanding the sequence, structure and functional space sampled by directed evolution. We have also extended these methods to allow the replacement of one trinucleotide sequence with another either predetermined (e.g. TAG) or random (e.g. NNN) sequence. This allows directed evolution to perform a “scanning mutagenesis” function and overcome the “codon bias” problems inherent with existing directed evolution methods. The transposon-based method has also been used to create new protein scaffolds by recombining two normally disparate, non-homologous proteins, so creating novel chimeric proteins.
Postgraduate Research Students
Miss Lisa Halliwell (Joint with Prof Jim Murray)
Mr Sam Reddington (Joint with Chemistry)
Drs Emyr Macdonald and Martin Elliot (Single protein molecule ET - School of Physics)
Dr Pierre Rizkallah (Protein structure determination – School of Medicine)
Dr Eric Tippmann (Non-natural amino acids - School of Chemistry)
Prof Rudolf Allemann (Protein molecular switches - School of Chemistry)
Dr Andrea Brancale (Molecular dynamic simulations – School of Pharmacy)
Prof Jens Ulstrup and Dr Qijin Chi (Single protein molecule ET - DTU, Denmark)
Dr Paul Barker (Protein engineering - University of Cambridge)
Dr Mark Howard (Protein NMR – University of Kent)
Prof Keith Wilson (Structural biology – York University)