The transition state regulators are DNA-binding proteins found in many bacterial species and are involved in the regulation of processes related to the organismâs defense. Each transition state regulator binds multiple DNA targets with affinity in the nanomolar range, in spite of not recognizing a consensus sequence. Structural studies of the N-terminal DNA-binding domain have revealed a mechanism by which the proteins bind DNA, however, to date no NMR structure of a full-length transition state regulator exists. AbrB is the most well-characterized transition state regulator, and homologs are found in both Bacillus subtilis and Bacillus anthracis. The sequences of AbrB from B. subtilis and B. anthracis are overall 85% identical, and understanding the sequence differences from a structural standpoint is key to understanding the function of each protein. The far-UV circular dichroism spectra of each were deconvoluted using a number of programs, and the estimates of secondary structure content compared to the predictions by PSIPRED. Multiple NMR experiments were performed in order to assign the backbone chemical shifts of AbrB from both B. subtilis and B. anthracis. Chemical shift analysis has recently proven to be a powerful tool to investigate protein structure, providing information on backbone dihedral angles, aromatic ring orientation and other parameters. A comparative analysis of the homologous proteins was undertaken by determining the change in chemical shifts (Îδ) of conserved residues. The Chemical Shift Index (CSI) was used to determine the secondary structure of AbrB based on chemical shift information. Results show that the N-terminal DNA-binding domain adopts the same structure as observed in the NMR structure of the isolated AbrB DNA-binding domain. The appearance of the TROSYs indicates that neither AbrBBA nor AbrBBS adopts a largely random coil structure, and likely contains the C-terminal α-helix predicted by PSIPRED.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-03252009-143618 |
| Date | 20 April 2009 |
| Creators | Perry, Nicole Alana |
| Contributors | William Miller, John Cavanagh, Dennis Brown |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-03252009-143618/ |
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