roduce forms of dihydrofolate reductases or DHFR that are 44 percent identical at the protein level. ITCHY or incremental truncation for the creation of hybrid enzymes was used to splice these DHFR enzymes together. Libraries of new and potentially interesting enzymes were created, but these new proteins do not necessarily have any enzymatic activity and therefore many of them were tested in the laboratory for activity.
Working on the computer, rather than in the laboratory, Manish C. Saraf, graduate student in chemical engineering and Costas D. Maranas, associate professor of chemical engineering developed FamClash to understand and predict which combinations of pieces from the original enzymes would cause clashes and diminish activity and which will form active hybrid enzymes.
"First we have the computer program generate all the hybrids that could form using ITCHY," says Saraf. "Then we look at every residue combination in each hybrid for pair clashes."
To function properly, protein strands need to fold in a specific way so that certain domains are next to or aligned with other domains. Both forms of enzymes studies here have similar structure and function, however, clashes occur in hybrids when they retain fragments from original enzymes that are not compatible with each other.
"Pairs of residues that are too big, or too small, or have the wrong electrical charge can cause these clashes that prevent these hybrids from folding correctly," says Saraf. "We hypothesize that the greater the number of clashes that exist in the hybrids, the less likely it is to fold correctly and therefore lower activity will be present."
The hybrid combinations are then ranked for predicted enzyme activity based on the number of clashes present.
"It is very helpful to experimentalists to know where introduced crossovers will produce high activity," says Horswill. "The long-term goal is to engineer enzymes for specific functions."
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