Protein Epistasis and the Biophysical Constraints on Catalytic Promiscuity

The limits of protein evolution are ultimately defined by biophysical constraints. Specifically, the maintenance of protein folding and stability is essential for the emergence novel enzyme function in the face of accumulating mutations. This fundamental requirement necessitates a viable mutational pathway towards beneficially altered function, but the frequency of such pathways has never been measured. To measure the biophysical constraints on the evolution of terpene biosynthesis, we performed a quantitative assessment of fold thermostability along all possible mutational pathways linking a wild type terpene synthase from Nicotiana tabacum (tobacco) to its catalytically evolved nonuple (9) mutant. We measured the thermal unfolding profiles of a 512 mutant (29 = M9) library using a high throughput fluorescencebased assay. These unfolding profiles revealed a broad range of stability phenotypes across the library. Co-variation analyses across the folding and stability landscapes of the 9 mutant combinations identified several statistically significant nonadditive effects and distinct thermal unfolding profiles. Together, these computational and experimental approaches uncovered additional levels of protein epistasis beyond the previously identified catalytic landscapes of the M9 library [1].

[1] O'Maille PE, Malone A, Dellas N, Andes Hess B Jr, Smentek, L, Sheehan I, Greenhagen BT, Chappell J, Manning G, Noel JP. Nat Chem Biol. 2008 Oct;4(10):617 – 23.