| When not infectious, virus particles offer scientists useful building blocks and display platforms with regular structures and large surface areas. The positioning of biologically active structures on the exterior surface, and payload molecules on the interior, allow chemists to synthesize large structures that impact biological systems in unprecedented ways. A complete understanding of any particle's native chemical reactivity is necessary to achieve control over the spacing and orientation of displayed structures. As a test case, the icosahedral Cowpea Mosaic Virus (CPMV), a plant virus with capsid structure known to near atomic resolution, was studied. There are no easily accessible cysteine residues on CPMV, so the genetic introduction of a surface cysteine residue should provide a uniquely addressable residue. A library of cysteine mutants was made and their reactivates surveyed. The local environment of the cysteine residue is critical to the reactivity and stability of the particles. The best behaved mutants were those in which the introduced thiol groups were sterically hindered enough to prevent inter-particle disulfide bond formation but open enough to allow to small molecule. Lysine side-chains were identified to react at similar rates to introduced cysteines with "thiol-specific" reagents, making completely selective labeling impossible in such polyvalent situations.;Incorporation of the unnatural amino acid azidohomoalanine was employed as an alternative strategy to generate uniquely reactive sites on a virus particle. Using the promiscuity of the methionine tRNA synthetase and a methionine auxotroph E. coli cell line provided by Prof. David Tirrell, the surfaces of bacteriophage Qbeta and Hepatitis B core particles were patterned with unnatural amino acids for the first time. Azidohomoalanine was chosen because the azide group is unique: it is highly energetic, yet unreactive with all functional groups in biology. Our group has developed catalysts for the completely selective cycloaddition reaction of organic azides with terminal alkynes, tailored for use in bioconjugation. The installation of azides into these particles under genetic control, followed by cycloaddition with alkynes of widely varying structure and complexity, comprises a two-step method for the incorporation of unlimited diversity in functional groups into precisely controlled positions on proteins and protein assemblies. |
