Stabilizing Protein Motifs with a Genetically Encoded Metal-Ion Chelator.
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. We demonstrate 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 a straightforward method for stabilizing coiled-coil structural motifs in recombinantly engineered proteins.
Department of Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Xiaozhou
X
Luo
Department of Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Tsung-Shing Andrew
TA
Wang
California Institute for Biomedical Research (Calibr), 11119 North Torrey Pines Road, La Jolla, CA 92307, USA.
Yong
Y
Zhang
California Institute for Biomedical Research (Calibr), 11119 North Torrey Pines Road, La Jolla, CA 92307, USA.
Feng
F
Wang
Department of Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; California Institute for Biomedical Research (Calibr), 11119 North Torrey Pines Road, La Jolla, CA 92307, USA. Electronic address: schultz@scripps.edu.
Peter G
PG
Schultz
D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry*; D020816:Amino Acid Motifs; D005296:Ferrous Compounds / Q000737:chemistry*; D005821:Genetic Techniques; D007477:Ions / Q000737:chemistry; D010455:Peptides / Q000235:genetics*; D055550:Protein Stability; D015032:Zinc / Q000737:chemistry*
D005296:Ferrous Compounds; D007477:Ions; D010455:Peptides; D015032:Zinc; D000409:Alanine