The Chan laboratory focuses on problems at the interface of chemistry and biology requiring the use of techniques spanning a wide range of disciplines. These include macromolecular crystallography, organic and inorganic synthesis, molecular cloning, protein overexpression and purification, and spectroscopy.

Fig. 1. Pyrrolysine structural and chemical biology. (A) Observed conformational changed of pyrrolysine following reaction with methyl-hydroxylamine. (B) Chemical synthesis of L-pyrrolysine.

Pyrrolysine Structural and Chemical Biology

Perhaps the most important result from the Chan laboratory has been the discovery of pyrrolysine, the 22nd genetically-encoded (it uses the UAG codon) amino acid from the crystal structure of monomethylamine methyltransferase, MtmB - in collaboration with laboratory of Prof. Joseph Krzycki (1) (Fig. 1A). Our current structural analyses suggest that pyrrolysine acts to bind methylamines and position their methyl groups in an orientation where it can be abstracted by a corrinoid cofactor upon docking of its cognate corrinoid-binding protein, MtmC. Our lab subsequently synthesized the amino acid (2) (Fig. 1B), and the Krzycki group used this amino acid to show that it could be incorporated into recombinant protein (3).

Current projects include the structure determination of additional pyrrolysine-containing proteins as well as proteins involved in pyrrolysine incorporation, and the development of practical methods to use pyrrolysine and pyrrolysine analogs for novel biotechnologies. Our first report detailing the use of this technology to site-specifically tag proteins has been recently published (4).

Fig. 2. Rh protein structure highlighting the channel, CO2 binding site, and residues proposed to link protein binding to the C-terminus to channel opening (5).

Membrane Protein Structure/Function

A relatively new focus of the lab has been the structural determination of membrane and membrane-inserting proteins. Our first success in this arena has been the structure of the N. europaea Rh protein (Fig. 2) in collaboration with Transmembrane Biosciences (TMB), a company in Pasadena, CA focused on developing novel membrane protein expression technologies. Like Amt proteins (ammonia channels), the structure of the N. europaea Rh protein revealed a homotrimer with a central substrate channel at the center of each subunit (5). The novel feature of the Rh protein structure was the presence of a cytoplasmic a-helix that oligomerizes to form a 3-helix bundle. Notably, a potential mechanism linking protein binding to the C-terminal helix to channel opening could be suggested from the structure. We are currently working to test this hypothesis by biochemical and structural methods.

Another major effort underway within this arena is the structural and biochemical study of Bacillus thuringiensis Cry toxins with Prof. Donald Dean (OSU). These proteins are soluble proteins that when activated insert into the membranes of insect midgut cells and promote insect death. We are interested in determining the structures of the membrane-inserted forms of these proteins, and in using this system to develop several novel biotechnologies.