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We have identified and characterized the first sRNA, DsrABb, in the Lyme disease spirochete, <em>Borrelia burgdorferi</em>; as well as, identified a non-canonical RNA chaperone Hfq. The alternative sigma factor RpoS (sigma 38 or sigma S) plays a central role in the reciprocal regulation of the virulence-associated major outer surface proteins OspC and OspA. Temperature is one of the key environmental signals controlling RpoS, but the molecular mechanism by which the signal is transduced remains unknown. DsrABb post-transcriptionally regulates the alternative sigma factor RpoS in response to an increase in temperature, associated with the tick to mammal transmission signal. A novel 5' end of the <em>rpoS</em> mRNA was identified and DsrABb has the potential of extensively base-pairing with the upstream region of this <em>rpoS</em> transcript. We demonstrate that <em>B. burgdorferi</em> strains lacking DsrABb do not upregulate RpoS and OspC in response to an increase in temperature, but do regulate RpoS and OspC in response to changes in pH and cell density. The 5' and 3' ends of DsrABb were mapped, demonstrating that at least four species exist with sizes ranging from 213 to 352 nucleotides. We demonstrate and characterize in vitro the interaction between DsrA and upstream region of <em>rpoS</em> mRNA. We hypothesize that DsrABb binds to the upstream region of the <em>rpoS</em> mRNA and stimulates translation by releasing the Shine-Dalgarno sequence and start site from a stable secondary structure. Therefore, we postulate that DsrABb is a molecular thermometer regulating RpoS in <em>Borrelia burgdorferi</em>.

Mass spectrometry has emerged as a powerful tool in the field of proteomics enabling characterization of proteins, their post translational modifications and determining protein-protein interactions. This versatile analytical technology allows both qualitative and quantitative measurements on a large number of proteins that is required to understand the complex molecular biology underlying the various cellular processes such as growth, development and response to stimuli. There are several mass spectrometry-based quantitative proteomic strategies reported in literature, all of which can be broadly classified into two groups: stable isotope-labeling approaches and label-free approaches. While the stable isotope labeling strategies are well established and applied extensively, label-free approaches are relatively new in the field of quantitative proteomics. The aim of this work was to evaluate the use of both labeling and label-free quantitative proteomic approaches with specific application to plant hormone signaling pathways. The use of stable isotope labeling for phosphorylation quantification was evaluated by the development and application of a new Phosphoprotein Acid cleavable Solid-phase Isotope-coded Reagent (PhASIR) to both model phosphopeptides and a phosphoprotein. The difficulties encountered with this approach such as complex labeling chemistries, presence of by-product, poor reproducibility and considerable loss of sample suggested that this approach was not suitable for quantitative phosphoproteomics. On the other hand, label-free quantification strategies are relatively simple, fast and can be easily applied to large-scale proteomic studies. A label-free, intensity-based quantitative strategy using a unique parallel fragmentation and a data-independent acquisition mode, termed LC/MSE was developed to carry out the functional analysis of the Arabidopsis thaliana receptor kinase BRI1 in vivo phosphorylation sites in response to brassinolide treatment. This label-free approach was also applied to carry out a comprehensive analysis of the changes in protein abundance in response to gibberellin treatment in Arabidopsis seedlings. The gel-based, LC/MSE approach termed GeLC/MSE allowed the simultaneous identification and quantification of over 200 gibberellin-responsive cytoplasmic proteins with atleast a greater than two-fold change in abundance. Overall, the LC/MSE approach proved to be a reliable method for label-free quantitative proteomics with the advantages of increased protein/proteome coverage, good analytical reproducibility and accurate quantitative results.

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.

Rapid and efficient methods for preparing isotopically labeled recombinant proteins and refining solved structures via NMR are presented. The former approach was developed for 2H/13C/15N isotopic labeling and specific protonation of the methyl groups of isoleucine, leucine, and valine (ILV) residues of rat brain calbindin D28K, a calcium sensor and buffer. This protocol produces cell mass using unlabeled rich media followed by exchange into labeled media at high cell density. Allowing for a short period for growth recovery and unlabeled metabolite clearance, the cells were induced. Additionally, the solution structure of LuxU, a subunit of the quorum sensing circuit of Vibrio harveyi, has been refined using residual dipolar coupling (RDC). In slightly anisotropic environments, large one-bond internuclear dipolar interactions no longer average to zero, and therefore can provide information on the average orientation of the corresponding vectors relative to the magnetic field. This ordering was induced by introducing Pf1 filamentous bacteriophage into a solution of 13C15N isotopically labeled LuxU.

The H-Ras protein is a GTPase important to cell cycle and differentiation. Mutations in this protein have been associated with 30% of cancers. A better understanding of this protein could lead to innovative treatments of cancers caused by the mutations. Solvent mapping of the crystallized Q61L mutant of the H-Ras protein with glycerol shows with potential areas of protein/protein interactions, areas that are of particular interest in the design of anti-cancer drugs. This study investigated whether glycerol can distinguish changes in the surface due to the mutation. A comparison was made between the wild-type and Q61L mutant H-Ras crystal structures in glycerol and aqueous solution. The structural analysis lead to the conclusion that the main changes observed were due to the solvent environment and not to the mutation.