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Article

Design of a Functional Protein for Molecular Recognition: Specificity of Ligand Binding in a Metal-Assembled Protein Cavity Probed by ¹⁹F NMR

Supporting Info

Allison J. Doerr, Martin A. Case,^† István Pelczer, and George L. McLendon*

Contribution from the Department of Chemistry, Princeton University, Princeton, New Jersey 08544

J. Am. Chem. Soc., 2004, 126 (13), pp 4192–4198

DOI: 10.1021/ja035798b

Publication Date (Web): March 10, 2004

†

Current address: Department of Chemistry, University of Vermont, Burlington, VT 05405.

In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

, glm@princeton.edu

Abstract

A metal-assembled homotrimeric coiled coil based on the GCN4-p1 sequence has been designed that noncovalently binds hexafluorobenzene and other similar ligands in a hydrophobic cavity, created by making the core substitution Asn16Ala ([Fe(bpyGCN4-N16A)₃]²⁺). The K_D of binding of hexafluorobenzene with [Fe(bpyGCN4-N16A)₃]²⁺ was observed to be 1.1(9) × 10^-⁴ M by diffusion NMR experiments. A control coiled coil with the core substitution Asn16Val ([Fe(bpyGCN4-N16V)₃]²⁺) exhibited a significantly weaker association with hexafluorobenzene, providing evidence that even in the absence of structural data, benzene-like ligands bind in the cavity created by the Asn16Ala substitution. ¹⁹F NMR was employed to observe hexafluorobenzene binding and to monitor titrations with competing hydrophobic and polar ligands similar in size and shape to hexafluorobenzene. All hydrophobic ligands bound with greater affinity than the polar ligands in the hydrophobic core, although the cavity seems to be somewhat flexible in terms of the sizes of molecules it can accommodate. Thus ¹⁹F NMR has proved to be a useful spectral tool to probe molecular recognition in a hydrophobic cavity of a metal-assembled coiled coil.