Thursday, August 22, 2013

Researchers use an unnatural amino acid to design a novel metal-binding protein

Someday designer metal-binding proteins could catalyze chemical reactions unseen in nature for industrial and medical applications. Unfortunately, these proteins are difficult to create from scratch. Now, using computational design, researchers have generated a novel metal-binding protein by incorporating an unnatural amino acid with a taste for metals (J. Am. Chem. Soc. 2013, DOI: 10.1021/ja403503m).
None of the 20 amino acids typically found in proteins has a strong affinity for metals, says Jeremy H. Mills, a postdoctoral researcher in the laboratory of David Baker at the University of Washington, Seattle. So, metal-binding sites in natural proteins often contain several amino acids working together to grasp a metal. Mimicking that precise orientation of several amino acids poses a serious challenge in the design of metal-binding proteins, Mills says.
Baker, Mills, and their colleagues sidestepped the problem using an unnatural amino acid called (2,2’-bipyridin-5-yl)alanine, or Bpy-Ala, which has micromolar affinities for a variety of metals. At first, the team attempted to design an easy-to-express enzyme that mimicked the activity of catechol dioxygenase. This enzyme breaks down the environmental pollutant catechol, but is difficult to express in laboratories, Mills says.
They used computer programs to redesign catechol dioxygenase’s metal-binding site to include Bpy-Ala. Then, they searched a database of protein structures for ones that express well in Escherichia coli and could spatially accommodate the redesigned metal-binding site. With some small structural tweaks, the researchers identified five promising protein sequences.
After they expressed those designed proteins in E. coli, the researchers tested their ability to bind iron via a spectroscopic technique. Two of the proteins appeared to bind the metal, but when the researchers solved the structure of one using X-ray crystallography, they did not see the metal-binding site. Instead, the part of the protein containing the Bpy-Ala amino acid had twisted so that the unnatural amino acid jutted into solution. With the unnatural amino acid in the wrong position, the protein couldn’t catalyze the desired reaction.
Disappointed, the researchers set a humbler goal: design a protein that just binds metals. This time, they instructed their computer program to place Bpy-Ala within a more rigid part of the protein, such as within an α-helix. Such structures, according to Mills, “are less free to move around,” and more likely to lock the Bpy-Ala into place.
The strategy worked. They identified one easily expressed protein with about 250 amino acids that could bind iron, based on its spectroscopic signature. The crystal structure of the new protein matched their computer design almost exactly. Also the novel protein bound cobalt, zinc, and nickel, with affinities in the micromolar to picomolar range.
The match between the team’s designed structure and the X-ray crystal structure is impressive, says Huimin Zhao of theUniversity of Illinois, Urbana-Champaign, as well as their use of an unnatural amino acid to create the metal binding site. The next step, he says, is to design a metalloprotein “that catalyzes a reaction that a natural protein cannot.”