On the structure and dynamics of cytochrome c3 and HU

Abstract

This thesis presents the results of research on the structure and dynamics of proteins us- ing NMR spectroscopy and molecular dynamics. The introductory Chapter 1 presents an overview of NMR and molecular dynamics methodology as applied to proteins and nucleic acids in solution. Chapter 2 presents the solution structure of cytochrome cg from Desulfomicrobium norvegicum (c3Dn). c3£)n is a tetrahaem protein involved in redox reactions in the bacterium. The oxidized and therefore paramagnetic isoform of c3Dn was assigned using standard sequential assignment methods. An ensemble of structures was cal- culated from restraints derived from NOESY spectra using the molecular dynamics program PARADYANA. The structure ensemble accurately represents the structure of the protein in solution as determined by the available data. The parameters of the para- magnetic tensors of the haem groups were fitted using calculated dipolar shifts and the X-ray coordinates. The NMR ensemble is also compared to the X-ray structure confirming that the structures in solution and in the crystal are highly similar. Chapter 3 presents the assignment of the HU protein from the thermophilic bac- terium Thermotoga maritima (HUrmar). This homodimeric protein is extremely sta- ble and acts as an architectural factor during the initiation of replication in bacteria. Unexpectedly for a thermostable protein WJTmar exists in two forms in solution as evidenced by the complete resonance assignment by triple resonance NMR techniques. The secondary and tertiary structure of form A resembles the crystal structure accord- ing to an analysis of chemical shift and NOESY data. Form B has very similar sec- ondary structure according to chemical shift data with the notable exception of the C-terminus. In contrast, the NOESY data for form B are much less complete indicat- ing a more labile structure. Chapter 4 examines the backbone dynamics of the two forms of liUTmar using NMR relaxation methods. Form A is found to have similar overall flexibility compared to the previously characterized HU protein from Bacillus stearothermophilus. Form B is shown to have a much higher degree of flexibility which is in agreement with the data from Chapter 3. The extent of this phenomenon is unusual for a thermostable protein and is discussed in reference to recent reports in the literature.

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