Texas A&M AgriLife research study to focus on amino acid radicals

A better understanding of these chemical reactions is likely to impact widespread disciplines, from the development of anticancer drugs to the sustainable production of solar energy to explaining how nonsteroidal anti-inflammatory drugs, or NSAIDs, such as aspirin and ibuprofen work in the body. However, amino acid radicals have been notoriously difficult to study.

“It is extremely difficult to experimentally solve the thermodynamic and kinetic redox properties of a single amino acid residue,” said Cecilia Tommos, professor in the department of biochemistry and biophysics at Texas A&M. “In addition, amino acid radicals are generally highly oxidizing and reactive species, further increasing the barrier for experiential characterization.”

To address these challenges, the Tommos research group has developed a library of well-structured model proteins that will allow detailed studies of the fundamental chemical properties associated with redox-active amino acids.

Tommos is the principal investigator of the study, which will be conducted in collaboration with other research groups at Yale University, the California Institute of Technology and Uppsala University in Sweden. Funding of $ 303,000 per year will be provided for the four-year grant, bringing the total to over $ 1.2 million.

Amino acid radicals and the enzymes that use them

Enzymes that use amino acid radicals are called redox proteins. These enzymes are essential for the functioning of many metabolic pathways in plants, animals and various microorganisms, Tommos said. Chemically, redox proteins and amino acid radicals enable a wide range of biochemical processes, including energy transformation, signal transduction, and DNA replication and repair.

Redox proteins use metallocofactors, organic molecules, and four types of amino acids – tyrosine, tryptophan, cysteine, and glycine – to perform electron transfer, ET, and proton-coupled electron transfer reactions, PCET.

“The four amino acids serve as essential one-electron redox cofactors, or radicals, in biocatalytic and multistep ET / PCET processes, some of which are essential to life on earth,” explained Tommos.

However, she noted that there was also a “sinister side” to these electron transfer reactions. When these reactions are induced by oxidative stress, they can cause significant cell damage.

A model system for studying tyrosine and tryptophan radicals

Tommos said the family of well-structured model proteins developed by his lab are specifically designed to study the formation, transfer and decay of tyrosine and tryptophan radicals.

“This model system allows us to study radical reactions of amino acids under fully reversible conditions in a structurally well-determined protein environment,” said Tommos.

She said the model proteins, called a3X proteins, were created by combining protein design with the site-specific incorporation of unnatural or non-canonical amino acids and detailed structural studies.

“A major breakthrough with the a3X model system approach has been achieved by the development of a very high potential protein film voltammetry method,” she said.

Protein film voltammetry is one of the techniques suitable for studying proteins that have electron transfer reactions, and is a method for studying the rates of chemical reactions catalyzed by enzymes.

Tommos said the unique properties of a3X proteins, combined with protein film voltammetry, enabled his research group to measure for the first time ever the potentials for reversible reduction of redox-active tyrosine and tryptophan residues.

“These findings also provided us with a critical springboard for what we wanted to accomplish with our next study,” she said.

Three specific objectives

Tommos said the three specific objectives of the current study will be to:

• Develop mechanistic and theoretical tools to study tyrosine / tryptophan-based multistage ET / PCET in model proteins and natural enzymes.
• Develop the model protein system and measure thermodynamic and kinetic parameters associated with multi-step tyrosine / tryptophan ET / PCET processes.
• Connect the mechanisms of tyrosine / tryptophan oxidation and radical decay to the dynamic properties of proteins.

“Each of these lenses is independent and will provide essential and unpublished information,” Tommos said. “If successfully completed, these specific goals will lead to a major advance in our understanding of amino acid radicals which, along with metals and organic redox cofactors, form the basis of biological ET and PCET. “