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Many Experiments:  Muratore Leads Amino Acid Research

Tim Borbet and Daniel Catt, chemistry research

Tim Borbet and Daniel Catt

Tucked away in a little corner on the south side of AU's campus is a vibrant biochemistry lab bubbling with activity. Under the guidance of Professor Kathryn Muratore, three pairs of students work on cutting-edge enzyme mutation projects.

Muratore wrote a computer program that identifies potential covariation in protein amino acid sequences and plans to test the effects these covariations may have on enzyme function through the use of mutagenesis and enzymatic assays.

Sam Sheftel, a first-year master's student in chemistry, and Shannon Christie, a senior majoring in environmental science, were the first two students to start working with Professor Muratore. Sheftel, who did his undergraduate work in biochemistry at AU, said he had "dabbled in programming" before and thought he should combine programming and biochemistry. Christie decided to try biochemistry research after taking a biochemistry class. Because she had no programming background, Christie had to teach herself how to code for computers, which she did by practicing and writing basic programs.

Sheftel and Christie's program analyzes sequences of amino acids in proteins in the C language. The C language is a general and older computer programming language. Sheftel and Christie are working on rewriting the program in Perl, which is designed for multitasking and real-time programming and has better memory allocation, as well as in Python. Because of this difference in language design, it would be difficult to simply "translate" the C language into Perl and Python. Christie looks at what the program does in the C language and then writes in the other languages for a similar action.  Sheftel and Christie hope to finish their work by this spring to write and publish a paper on their work.

The computer program produces potential covariation in protein amino acid sequences, but what the program produces needs to be experimented on to figure out whether it has real-world application. Daniel Catt, a senior premed student, and Tim Borbet, a senior biochemistry major, are in the first stages of the experimental process.

Conceptually, the active site of an enzyme has a certain amino acid sequence that determines what the enzyme can act on—an aspect of the enzyme called its specificity. Catt and Borbet are trying to determine exactly how specific the enzyme is. After this is determined, they can then move on to attempting to mutate the enzyme to change its specificity and try to make the enzyme act on more than one substrate. Currently, both are performing protein purifications, meaning they are working with the actual enzyme and have not started mutations.

Catt and Borbet say that they enjoy their work but it does come with some challenges. The work is very tedious because they have to perform the same tasks multiple times to prove consistency. However, getting the conditions right for the purification, mainly in regards to the pH levels, has proven to be the biggest challenge so far. Their goal is to get three consistent trials so they can publish their work.

Tamra Fisher, a junior biochemistry major, and Jennifer Gaston, a senior psychology major and chemistry minor, are further along in their research. They have moved past the purification stage and have started mutations on the enzyme malate dehydrogenase.

Malate dehydrogenase is the enzyme that acts on malate, the compound that gives green apples their sour taste. Fisher and Gaston's procedures involved the same steps that Catt and Borbet took, and having already confirmed the enzyme's substrate, they are focused on modifying its actions to accommodate lactate, a derivative of malate.

Adapted from "One Biochemistry Lab, Many Experiments" by Vijaya Singh, Catalyst, Winter 2011.