Institute for Protein Design

A Computationally Designed Metalloprotein Using an Unnatural Amino Acid

February 14, 2014    Mills, J. H., Khare S. D., et al. (2013).  Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy.  Journal of the American Chemical Society. 135(36), 13393-9.

What if scientists could design proteins to capture specific metals from our environment?  The utility for cleaning up metals from waste water, soils, and our bodies could be tremendous.

Computational Metal Binder Design

Dr. Jeremy Mills and collaborators in Dr. Baker’s group at the University of Washington’s Institute for Protein Design (IPD) address this challenge in the first reported use of computational protein design software, Rosetta, to engineer a new metal binding protein (“MB-07”) which incorporates an “unnatural amino acid” (UAA) to achieve very high affinity binding to metal cations.

Mills et al report their work in a paper entitled Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy, published in August 2013, in the Journal of American Chemical Society.

Mulligan metal binder figure

The designed metalloprotein MB_07 (ribbon) bound to a metal ion (sphere). Key binding site residues (sticks), including the primary ligand UAA (chemical structure shown in lower left), are shown coordinating the metal cation. Illustration rendered by Vikram Mulligan.


With few exceptions, naturally occurring proteins are constructed from only 20 amino acids. However, recent technological advances have afforded researchers the ability to genetically encode amino acids that do not exist in nature, UAAs, into naturally occurring proteins.  The UAAs are used to enhance, alter, or study protein functions. For example, UAA side chains can be incorporated into proteins to serve as orthogonal reactive groups to include elements such as fluorescent probes, DNA conjugates, and a host of posttranslational modifications — a characteristic otherwise not afforded by the canonical 20 amino acids.

The UAA used by Mills et al  is (2,2′-bipyridin-5yl)alanine, or “Bpy-Ala” which has the ability to bind a variety of di-valent metal cations.  The remainder of the computationally defined metal binding site is constructed from the 20 native protein side chains. This binding site, in addition to the UAA, greatly increases the metal binding affinity of the designed protein.

This new metalloprotein has been shown to tightly bind many biologically relevant metal ions including zinc, iron, nickel, and cobalt, as well as some metals that occur less often in nature like palladium.


 IPD researchers have shown that computational protein design around a UAA will allow for the generation of a number of novel proteins including new metalloproteins which may one day be used to sequester or re-capture toxic and precious metals.

Why Is This Important?

Proteins that require a metal ion cofactor, metalloproteins, make up close to half of all naturally existing proteins. Metalloproteins range in function from facilitating storage and transport processes in the cell to catalyzing nitrogen fixation and molecular oxygen reduction to mediating signal transduction. Given their prevalence, functional design of novel metalloproteins will both provide a better understanding of how they work and result in the development of protein tools that have therapeutic, biotechnological, and environmental applications.

A designed metalloprotein, such as MB_07, that has high affinity for specific metal ions may have a strong environmental impact as an integral reagent in removing toxic and radioactive materials from wastewater streams. This metal-scavenging activity could also be advantageously employed in cases such as blood detoxification by efficiently titrating out and sequestering the toxic culprit. Furthermore, the design of metalloproteins with new catalytic activities (metalloenzymes) would facilitate the exploration of more efficient, cost-effective, and environmentally friendly alternatives to catalysts currently used in many synthetic and industrial chemical reactions.

What’s Next?

The successful results described here highlight the potential to design proteins of new functions around unnatural amino acids of varying structure and function. This ‘bottom up’ approach should facilitate the design of a number of new proteins with exciting properties that would be difficult, if not impossible, to achieve using naturally occurring amino acids.

This article was authored by Dr. Ratika Krishnamurty, Technical Writer  ( at the Institute for Protein Design, with kind input and guidance from the researchers noted in this article.