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Volume 88 Issue 41 | p. 10 | News of The Week
Issue Date: October 11, 2010

New Window On Phosphorylation

Protein Chemistry: Structural mimics provide a handle on tough-to-capture modification
Department: Science & Technology
Keywords: proteins, antibodies, posttranslational modification, phosphorylation
8841NOTW7200_page10
 

For the first time, stable analogs of phosphohistidine have been used in peptide and protein synthesis and in antibody production. These analogs give researchers a new way to probe histidine phosphorylation, a common protein modification that helps control enzyme activity in cells.

Phosphorylation is the addition of phosphates to hydroxyl groups on serine, threonine, and tyrosine residues and to imidazole nitrogens on histidine. But histidine phosphorylation “has been a bit of a blind spot for chemists and biochemists because of the intrinsic instability of the modification,” says Tom W. Muir, a professor at Rockefeller University and leader of the new study.

Muir and postdoc Jung-Min Kee developed stable analogs of phosphohistidine that don’t hydrolyze or isomerize and used them to synthesize modified peptides and proteins (J. Am. Chem. Soc., DOI: 10.1021/ja104393t). The phosphoryltriazolylalanine-based analogs mimic the geometry and electronics of phosphohistidine but replace the N–P bond of the natural amino acid with a nonhydrolyzable C–P bond. “That nitrogen-phosphorus bond is extremely labile,” Muir says. “It’s a liability in terms of biochemical analysis. It had to go.”

Muir and Kee collaborated with Bryeanna Villani and Laura R. Carpenter of cell-biology firm Active Motif in Lake Placid, N.Y., to raise antibodies against peptides containing the analogs. “Prior to this study, there were no antibodies that specifically recognized phosphohistidine in any context,” Muir says. The new antibodies specifically recognize histone proteins with phosphorylated histidines but not their nonphosphorylated or nonhistidine phosphorylated counterparts.

The antibodies will allow researchers to “address a series of critical issues regarding the evolution and function of protein histidine phosphorylation in mammalian systems that have languished in the literature for the past 25 years,” says Melvin I. Simon, an emeritus professor of biology at California Institute of Technology.

The current antibodies recognize phosphohistidine only in the context of histone tail peptides, but Muir’s group is developing others that will recognize phosphohistidine in any context. “Such reagents would be incredibly powerful in proteomic studies for looking at broad occurrences of this modification,” Muir says.

 
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