Essentially all aerobic organisms require trace amounts of copper to function; however, when it is not properly regulated copper becomes a toxin and, therefore, strict regulation of copper homeostasis is vital. Surface cell transporters, such as Ctr1, are responsible for the uptake and transport of copper ions but the mechanism of copper delivery, dependent on metallochaperones, is still debated. Generally copper metallochaperones contain thiol-rich binding sites. Previously our lab has had great success studying heavy metal binding (Pb(II), Cd(II), Hg(II), As(III), etc…) in a three-fold symmetric thiol-rich environment using de novo designed three-stranded coiled coils (3SCC). K-edge X-ray absorption spectroscopy of Cu(I)-bound TRIL2WL16C (Sequencce: G-LKALEEK&lt;span style="font-size:10.8333px; line-height:17.3333px"&gt;4&lt;/span&gt;-G) at multiple pH conditions has revealed a heavy back-scatterer consistent with a short Cu-Cu distance of 2.66 Å with mixed two- and three-coordinate Cu-S environments. Additional analysis of the Cu-S charge transfer band via UV spectroscopy revealed a binding stoichiometry of two copper ions to one 3SCC. These data support a model where a binuclear Cu(I) site coordinated with multiple sulfur atoms exists in this system, much like Cu(I) binding to the repressor CopY.1 Ag(I) is often used as a probe for metallochaperones and similarly we have examined the K-edge EXAFS of Ag(I) binding to TRIL2WL16C. As observed in CopY, we see a two- or three-coordinate cysteine-bound Ag(I) ion but no Ag-Ag interaction is observed.1 Therefore, we conclude that the TRIL2WL16C series produces similar metal binding stoichiometries for both Cu(I) and Ag(I) as in the native repressor, CopY. 1) Cobine, P.A.; George, G.N.; Jones, C.E.; Wickramasinghe, W.A.; Solioz, M.; and Dameron, C.T. (2002) Biochemistry, 41, 5822-5829.

De novo designed metallopeptides: An unexpected binuclear Cu(I) site

251st National Meeting (2016)

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