SecA is a 100kDa ATPase that interacts with SecYEG, the transmembrane channel, to drive transport of preproteins designated for secretion. Despite considerable efforts to parse the individual steps required for protein translocation, a detailed and conclusive understanding of this process remains obscure. To explore the role of ATP hydrolysis in controlling the global conformational reaction cycle, a mutation was designed in the catalytic base of the SecA active site. Surprisingly, we found that a single charge change in the active site leads to intriguing changes in the thermodynamic behavior of SecA, indicating that electrostatics in the active site are allosterically coupled to global conformational changes in SecA. Additionally, active site electrostatics are shown to control two separate conformational pathways in SecA that govern the reaction cycle of this mechanoenzyme. The equivalent mutation in a model DEAD-box helicase produced the same effects, illustrating that the delicate balance of electrostatics in the active site and the coupling of this balance with global protein dynamics could be a general feature of the helicase family. This balance is important in many stages of the conformational reaction cycle, playing a role in nucleotide release rate, the rate of the total reaction, and controlling the global conformation of the enzyme. In this thesis, we present a model in which a network of electrostatic interactions in the active site, modulated by bound nucleotide, controls the global conformation of SecA via cascading order-disorder transitions. We propose that this behavior is a general property of helicase motors, and that this novel allosteric mechanism is distinct from the structurally related ABC Transporter family.
|Advisor:||Hunt, John F.|
|School Location:||United States -- New York|
|Source:||DAI-B 70/12, Dissertation Abstracts International|
|Keywords:||ATPase, Allostery, Conformational change, Electrostatic control|
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