Stabilizing Protein Motifs with a Genetically Encoded Metal-Ion Chelator

Abstract

The N-peptide of HIV gp41 forms a trimeric coiled-coil intermediate during host cell-viral fusion. Stable mimics of this coiled coil could potentially serve as HIV vaccine candidates or inhibitors of viral entry. Therefore, a variety of approaches have been investigated to maintain the N-peptide in its trimeric helical conformation. Here, we utilize a genetic method to incorporate the metal chelating noncanonical amino acid (2,2'-bipyridin-5-yl)alanine (BpyAla) into IZN17, an established trimeric coiled-coil gp41 model. Wedemonstrate that BpyAla-IZN17 acquires Fe(II) during expression in Escherichia coli, resulting in the formation of a highly stable IZN17 helical trimer with a Tm > 95°C. Removal of Fe(II) results in a 27°C decrease in thermal stability. Replacement of Fe(II) with Zn(II) also affords a Tm > 90°C. Thus, the genetic introduction of BpyAla into polypeptide provides astraightforward method for stabilizing coiled-coilstructural motifs in recombinantly engineered proteins.