The research conducted by the group centers on the protein folding and subunit assembly processes. The bacterial luciferase heterodimer is our model enzyme, which provides an assembly reaction resulting in the emission of visible light. The research uses methodologies derived from chemistry and biology, including mutation of individual proteins, molecular modeling, enzymology and bio-luminescence. Currently, we have a variety of active research projects in the lab. For details of individual research projects undertaken in the laboratory, please see below.
Engineering protein biosensors: Looking at fusion proteins
The detection of many biological materials has an important role in a wide range of applications, including clinical diagnosis, forensic analysis, environmental monitoring, and anti-terrorism. This has motivated a significant interest in the further development of convenient biosensors, which offer the potential to replace traditional biochemical assays in applications. Creating a luciferase fusion protein that will function as a biosensor is a real possibility. The expression of the enzymes a and b subunits as a single polypeptide chain can be generated by inserting an in-frame linker. The length and structure of the linker can be manipulated to allow the subunits to interact normally and allow functionality (light production) or not. Specific protein motifs e.g. Zinc fingers can be included within the linker for targeted applications.
Role of strategic residues in protein subunit interfaces
Bacterial luciferase is a light emitting enzyme predominately found in bioluminescent sea creatures. The functional protein is composed of two subunits, the a and b. An interesting characteristic of luciferase is that it is able to assume two native conformations: the active heterodimer as detailed above and the inactive kinetically trapped homodimer composed of two b subunits. It is suggested that the increased stability of the b homodimer lies in the subtle differences at the subunit interace of this protein and the heterodimer. A solvent accessible cleft observed within the core of the heterodimer interface is absent within the homodimer, perhaps allowing for additional electrostatic interactions and salt bridges to occur. Introducing point mutations into this region in an attempt to create an artificial cleft within the beta homodimer may highlight strategic residues and interactions providing the increased stability. (Daniel Martinez)
Cyanogen modification to elucidate essential residues
Cyanogen (Ethanedinitrile; C2N2) readily converts both inter- and intra- molecular salt bridges into covalent bonds. Using this reagent with bacterial luciferase from Vibrio harveyi the importance of intramolecular associations as they relate to enzyme function in vitro may be examined. The reaction of luciferase with cyanogen was seen to result in a rapid loss of activity. The inactivation appeared to be the result of modification of the a subunit, resulting in several newly formed species. MS revealed three solvent accessible ion pairs that had been crosslinked. One ion pair contained residues highly conserved between homologues is seen to have no tolerance for mutation. We suggest that cyanogen inactivation has detected critical bonds in or near the active center of bacterial luciferase. (Zachary Campbell)
Roles of non-prolyl cis-trans isomerization reactions
X-Pro peptide bonds are often in the cis conformation in native proteins. X-Pro dipeptides exist essentially completely in either the trans or cis forms in native proteins but as an equilibrium mixture of these forms in unfolded proteins. The cis/trans isomerizations of such bonds determine the rate of some slow folding reactions in vivo. Cis peptide bonds, which do not involve prolines, have been found in folded proteins, but they are extremely rare, because the cis form of a non-prolyl peptide bond is intrinsically unstable. It is suggested that the trans isomer is favored over the cis in the absence of residual structure. The recognition and assignment of peptide bond isomerization reactions are crucial to the development of a folding mechanism and to understanding the role of the amino acid sequence in directing the folding reaction. In the a subunit of luciferase, b-strand 3 terminates with a bulge that protrudes into the core of the barrel. This bulge contains a cis peptide bond between residues Alaa74 and Alaa75. Alaa74 and Alaa75 form the bottom floor at the entrance of a small cavity projecting off the larger and deeper pocket at the centre of the b barrel of the a subunit. The likely involvement of the isomerization of this highly conserved peptide bond (in most members of the bacterial luciferase family) in contributing to the folding mechanism of the bacterial luciferase, motivated for an intense investigation. (Judith Hornby).
Protein moieties targeted in temperature-sensitive refolding
Earlier work in this lab highlighted a temperature sensitive folding phenotype within luciferase. The phenotype came about due to an interesting mutation from a set of C-terminal deletions of the b-subunit of bacterial luciferase. Normal levels of the thermally-stable luciferase activity is monitored when the bacteria is grown between 18-20oC. However when the same cells are grown between 25-37 oC, the yield of active luciferase is severly compromised. It is thought that this region of the b-subunit and particularly residue 313 is not important for the structure or stability of the folded luciferase, but must be involved in the folding reaction of the protein. (Judith Hornby)
Use of NMR assignments to investigate disordered regions within proteins
When X-ray elucidation of a protein reveals little to no electron density within a region of a protein, this region is thought to be unstructured. In the case of luciferase, the area of interest consists of residues 262-290 of the a subunit. This region is near the active site pocket of the enzyme and is necessary for a high quantum yield reaction suggesting it undergoes a structural change upon substrate binding. Chemical shifts of labeled amide and a carbons can be used in conjunction with TROSY-related NMR experiments to signal a shift from an unstructured to a structured region suggesting essential functions to structural moieties. (Judith Hornby).
