| Department of Chemistry | Faculty of Science

Proteins: Folding, Structure, Function and Dynamics
We are investigating the relationships between protein structure, function, folding and dynamics, at atomic resolution. We use a multidisciplinary approach involving experimental techniques: multi-nuclear heteronuclear NMR spectroscopy, optical spectroscopies, differential scanning calorimetry, stopped-flow rapid mixing, computational techniques: bioinformatics, and molecular dynamics, while using molecular biological techniques to overexpress and modify proteins of biological and medical importance. Current projects are described below.

Hisactophilin: Model Beta-Trefoil Protein

Hisactophilin is an actin-binding protein from the slime mold, Dictyostelium discoideum. The structure of this protein is representative of the beta-trefoil superfold, an intriguing all-beta structure adopted by a group of proteins with unrelated sequences and functions. Additionally, hisactophilin is co-translationally modified by N-terminal myristoylation, which has been shown to be important for pH sensitive membrane binding in which the myristoyl group moves from a buried position to one accessible to the membrane.

We are conducting in depth investigations of the stability, folding, and myristoyl switching of hisactophilin using NMR spectroscopy, optical spectroscopies, chemical and thermal denaturation techniques and stopped-flow kinetics, in combination with molecular dynamics and computational modeling.

Myristoylated hisactophilin is shown above, with the myristoyl group as red spheres in the buried position, and several important mystoyl binding locations that are studied in our lab through biophysical analysis of mutations at those postions shown as colored spheres: F6 in orange, V36 in yellow, L76 in green, I85 in cyan, I93 in blue, and I118 in purple.

ThreeFoil: A Rationally Designed Symmetric, and Carbohydrate Binding Beta-Trefoil

The beta-trefoil superfold displays three-fold structural symmetry, but at a sequence level this symmetry is low to undetectable for most members. Several studies had suggested that the beta-trefoils might have arisen from the repetition of a single subdomain unit (one-third of a beta-trefoil). In order to demonstrate and explore the validity of this model we reconstructed the perfectly symmetrical ThreeFoil, using bioinformatics and computational modelling techniques.

Not only does ThreeFoil display remarkable symmetry, it has high thermal stability, unfolding at ~95 degrees Celsius, unfolds very slowly, with an unfolding half-life on the order of years, and binds to multivalent carbohydrates.

ThreeFoil is shown above with it's symmetric carbohydrate ligands as light green sphers (with coordinating sidechains as sticks), structurally important buried water molecules in light blue, and a metal ion that binds along the axis of symmetry shown in yellow (with coordinating sidechains as sticks).

Superoxide Dismutase: Does Misfolding Cause Disease?

Familial Amyotrophic Lateral Sclerosis (fALS) is a neurological disorder characterized by rapid degeneration of motor neurons, progressive paralysis and inevitably death. Mutations in the metalloenzyme, Cu,Zn-Superoxide Dismutase are associated with ~20% of all fALS cases, and a prominent disease hypothesis is that aggregation of mutant SOD is neurotoxic. However, the mechanism by which mutant SOD aggregates is unknown. Furthermore, because Cu,Zn-SOD undergoes various stabilizing post-translational modifications (metal binding, disulfide bond formation, and dimerization), the form of SOD that is relevant to ALS toxicity is unknown.

Using a number of biophysical techniques, we investigate how mutations affect the stability, folding behaviour and aggregation tendency of various forms of Cu,Zn-SOD. As many forms of Cu,Zn-SOD may be relevant to ALS toxicity, this work is important for unraveling the complexity of Cu,Zn-SOD-linked fALS.

Cu,Zn-SOD is shown above in the native dimer form, with the various mutations studied in our lab shown as yellow sticks. Copper and Zinc shown as orange and black spheres, respectively.

Research