19F NMR studies on DnaB helicase
Date
2014
Authors
Kuppan, Kekini Vahini
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NMR (nuclear magnetic resonance) spectroscopy is an important technique used for structural characterisation of proteins under near-physiological condition. It is used for determining the structure of proteins at atomic resolution and to study protein-protein interactions. However, its major limitation is the size of protein. The limitation can be overcome by solution state fluorine NMR, a promising technique which is used for studying the structural dynamics of large proteins by selective fluorine labelling. Cellular processes such as bacterial DNA replication is carried out by large protein complexes such as DnaB helicase, DnaG primase and DNA polymerase enzyme. It is necessary to study these proteins under physiological conditions to gain insights into the process. DnaB is primary DNA helicase in the replisome and its main function is unwinding the duplex DNA. It interacts with various other proteins and performs different functions during the process. Its structural characterisation by electron microscopy has established that it adopts two different rotational symmetry states (C3 and C6) and the conformational interchange occurs in N-terminal domain only. The symmetrical states were determined under non-physiological conditions. The factors triggering the conformational change were determined under physiological conditions however; the precise conformation adopted by DnaB under these conditions is elusive. To gain insights into structure, dynamics and interactions of DnaB under physiological conditions, I have studied the DnaB helicase and partner proteins by fluorine NMR spectroscopy. In this thesis, I report my studies that employed fluorine 1D NMR to study conformational changes of hexameric DnaB helicase with mass of 315 kDa in solution under physiological conditions. Trifluoromethylphenylalanine (tfmF) was the chosen fluorine label, incorporated into DnaB site-specifically. DnaB helicase and their partner proteins from Escherichia coli and Bacillus stearothermophilus were studied to observe the significant features in both systems. tfmF labelled E.coli DnaB was expressed by cell-free protein synthesis and examination at different pH established that its N-terminus is flexible in solution under near-physiological conditions rather than adopting rigid conformation. Furthermore, the study revealed that its interaction with helicase loader induced more flexibility into N-terminus of DnaB. The complex of E.coli DnaB and its helicase loader is a 480 kDa protein complex and the presented fluorine data are first attempts to study the conformational changes in such large protein systems. Bacillus stearothermophilus DnaB helicase was studied with its primase, helicase loader and magnesium ion. TfmF was incorporated into DnaB by in vivo method. The fluorine data showed that N-terminus is flexible similar to E.coli DnaB. However, its interaction with primase induced rigidity to N-terminus and adopted C3 symmetry, which is in concurrence with previous work. Moreover, the gel filtration data showed that magnesium ion rendered the integrity of hexamers by forming unstable monomers. We report the initial studies on Bacillus stearothermophilus helicase loader, DnaI. The fluorine NMR and gel filtration data suggests that it interacts with DnaB monomer instead of hexamers. The presented data shows fluorine NMR as a useful tool in determining the structural dynamics of large protein systems in solutions and its data can supplement previous structural information.
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