Structural Biology Group, KEK, PF* University of Zurich** University of California Santa Barbra*** HHMI, University of Oregon****
○Mohammad S Yousef* Nicole Bischoff** Collin Dyer*** Walt Baase**** Brian Matthews****
We have designed a molecular switch in a T4 lysozyme construct that controls a large-scale translation of a duplicated helix. As shown by crystal structures of the construct with the switch on and off, the conformational change is triggered by the binding of a ligand (guanidinium ion) to a site that in the wild-type protein was occupied by the guanidino head group of an Arg. In the design template, a duplicated helix is flanked by two loop regions of different stabilities. In the "on" state, the N-terminal loop is weakly structured, whereas the C-terminal loop has a well defined conformation that is stabilized by means of non-covalent interactions with the Arg head group. The truncation of the Arg to Ala destabilizes this loop and switches the protein to the "off" state, in which the duplicated helix is translocated approximately 20 Angstrom. Guanidinium binding restores the key interactions, restabilizes the C-terminal loop, and restores the "on" state. Thus, the presence of an external ligand, which is unrelated to the catalytic activity of the enzyme, triggers the inserted helix to translate 20 Angstrom away from the binding site. The results illustrate a mechanism for protein evolution in which a sequence duplication followed by a point mutation can lead to the establishment of new functions. Moreover, a fluorescence-based optical method was developed to detect the ligand-triggered helix translation in solution.
(Yousef et. al, 2004,2006)