(169a) Understanding the Reaction Mechanism of Protein Splicing in Order to Gain Insights for the Engineering of Inteins

Garde, S., Rensselaer Polytechnic Institute
Shemella, P., Rensselaer Polytechnic Institute
Nayak, S., Rensselaer Polytechnic Institute
Belfort, G., Rensselaer Polytechnic Institute
Pereira, B., Rensselaer Polytechnic Institute
Derbyshire, V., Wadsworth Center, NYS Department of Health
Van Roey, P., Wadsworth Center, New York State Department of Health, and School of Public Health, University at Albany
Belfort, M., Wadsworth Center, NYS Department of Health

Inteins are proteins within proteins! They are intervening polypeptide sequences that undergo self-splicing to ligate the exteins (the external protein fragments) in order to yield a functional host protein. The intein reactions are complex, stepwise, and time coordinated. They can be controlled or isolated by performing appropriate mutations at the N and/or C junctions. As a result, inteins have become something of a workhorse in protein manipulations and in novel biotechnology applications including protein purification. Their role in these applications can be adjusted or improved through rational engineering of the intein, but such engineering requires a more detailed understanding of the intein reaction mechanism.

Inteins naturally present excellent systems for the development and application of modeling approaches focused on understanding enzymatic reactions. The ability to perform complementary molecular biology and biochemical experiments can provide additional insights as well as confirm predictions of the modeling effort. We will present our ongoing work on understanding the reaction mechanisms of inteins and how it pertains to the engineering of inteins. Our work uses classical molecular dynamics, ab-initio quantum calculations, as well as systematic site directed mutagenesis experiments.