Jones, D. Dafydd, Dr

Dr D. Dafydd Jones - PhD

Construction of artificial protein scaffolds that act as molecular switches

The ability to design molecules that can change their properties in response to a desired input will allow significant new possibilities for creating novel sensors, modulators and transducers for use in both natural and artificial contexts. The capacity of proteins to fulfil the function of a molecular switch is well established in nature, as demonstrated by their key roles in regulating many biological processes. While it might appear easiest to modify natural protein switches, they have evolved to fulfil specific requirements within a certain biological context, making them difficult to adapt for new applications. As a general approach for creating tailored protein switches, we couple the structures and hence functions of normally unrelated proteins through signal-dependent conformational changes so that the output is modulated by a desired input. In order for the protein to act as a switch, existing proteins will require radical redesign or even the creation of new scaffolds but with natural proteins providing the inspiration and guidance for their construction. Both rational protein engineering and a recently developed transposon-based non-homologous directed evolution method are used to construct these artificial protein switches. 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.

We are also interested in using proteins as novel current modulators and transistors for use in nanoelectronics.

(a) An artificial DNA binding cytochrome. (b) Controlling enzyme (TEM-1) activity through linked conformational changes in a sensing domain (cyt b 562).

Engineering proteins using an expanded genetic code.

In collaboration with Dr Eric Tippmann in Chemistry, we are exploring use of engineering proteins through expansion of the genetic code. 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 unnatural 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.

The influence of indels on protein structure, function, folding and misfolding.

The general accepted model is that the insertion or deletion of an amino acid from the protein backbone will compromise the stability of a protein and hence disrupt function. However, this model is over-simplistic as indel events form an integral part of the evolutionary process. Therefore the general rules for the consequences of indel mutations are not fully understood. We are using the model proteins TEM-1 β -lactamase and the lipoic acid binding (lipoyl) domain to help us understand the consequence of indel mutations on protein structure, function, folding and misfolding so providing us with further insights into the structural plasticity of proteins. We have discovered that deletion of a single amino acid can have beneficial effects, resulting in substantially enhanced activity of TEM-1 towards normally poor substrates. Removal of residues from the lipoyl domain causes the protein to access a misfolded dimeric conformation.

Movie showing the amino acids (green balls) in TEM-1 that are tolerant to an amino acid deletion. The blue residue is the active site serine.

Structure, function and folding of novel intracellular subtilisin serine proteases

During the course of evolution, proteins with homologous structures have diverged to adapt their structure and function under a particular selection pressure but generally the folding pathway and mechanism has been conserved. But can the folding pathway itself be the subject of evolutionary pressure and can this in turn influence the functional properties of the protein?

We are interested in determining the properties of a novel class of intracellular subtilisin (ISP) serine proteases. The more commonly studied homologous extracellular subtilisins (ESPs) require a prodomain to act as molecular chaperone to fold the mature protein. The ISPs lack the prodomain and so fold via a different pathway to reach a native state structurally homologous to the ESPs. However, the ISPs are dimeric and they contain a 16-20 amino acid N-terminal extension that appears to be important for the structure and activity of the proteins. We will use the ISPs as a model system to understand how changes to the primary structure can affect not only protein function and structure but to also examine how such mutations can alter the folding pathway by which a native protein is reached.

Primary structure differences between extracellular and intracellular subtilisin serine proteases.

Technology developments – transposon-based methods for directed evolution

We have also developed a range of transposon-based technologies that 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 will alter the structure and hence function of protein in ways not accessible to substitution mutations alone thus expanding the sequence space that can be sampled by directed evolution. We have also extended these methods to allow the replacement of one trinucleotide sequence with another either predetermined or random (e.g. NNN) sequence. This will allow 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 recombine two normally disparate, non-homologous proteins, so creating novel chimeric proteins.