Purification and Preliminary Characterization of the TM0727 Protein from Thermotoga maritima

The TM0727 gene of Thermotoga maritima is responsible for encoding what has been reported to be a modulator of DNA gyrase (pmbA). Although the function of pmbA is still unknown, it is believedto be involved in cell division, carbon storage regulation, and the synthesis of the antibiotic peptide microcin B17. It is suggested that it serves together with tldD, a known zinc dependent protease, tomodulate DNA gyrase. TM0727 is believed to be a zinc dependent protease that binds zinc in the central active site of the molecule, located between two equivalent monomeric units. However, thecrystal structure determined by Wilson et al. (2005) did not contain zinc. It therefore remains to be seen if TM0727 requires zinc for activity, or regulation, and if the protein is indeed a protease. To begin studying this protein, the gene was expressed in BL21(DE3) pLysS cells and the induction time was optimized. Using affinity and ion exchange chromatography, the protein has been successfully purified. The purification procedure can be replicated to obtain sufficient protein for characterization. Purification results show that the protein loses stability after 24 hours and remains stable under an imidazole-free lysis workup. Preliminary characterization of TM0727 has focused on understanding the protein’s structuralproperties through tryptophan fluorescence anisotropy measurements. The four tryptophan residues located within the TM0727 dimer fluoresce at different maximum wavelengths and with differentintensities upon excitation with 295nm light. These emission properties are highly sensitive to the environment (solvent, surrounding residues) of each tryptophan residue. The low number oftryptophans allows for a specific monitoring of the protein’s structure as it denatures. As more denaturant is added to the protein, its tryptophan environments have clearly altered. This is indicative of unfolding and increased solvent exposure of the protein. This unfolding has been confirmed with the addition of a fluorescent quencher. Additionally, fluorescence anisotropy measurements have been carried out on the protein to gain a preliminary understanding of the rotational dynamics of the tryptophan residues. These experiments excite the tryptophan residues within the sample using a polarized light source. Polarized emission is then detected, the degree of which depends on the rotational dynamics and local environment of the tryptophan residues. The protein was denatured and the changes in emission were recorded to detect these structural changes. Results have shown a large change in quaternary structure, consistent with a dimer to monomer transition, occurs at 1.5M Guandidine HCl. There has also been an examination of the crystal structure for the location of a potential active site. The inner cavity of the protein was inspected visually to locate a potential location for a catalytic triad, specifically the amino acids found in the active sites of serine, cyteine, and aspartateproteases. It was found that a potential aspartic protease active site may be located between the Asparate286 and Aspartate287 residues. Further investigation is warranted to test this remotepossibility.

A Computational Analysis of the Collision-Induced Dissociation Mechanisms of the Dipeptide Glycine-Serine Under Mass Spectrometry

Collision-induced dissociation (CID) of peptides using tandem mass spectrometry (MS) has been used to determine the identity of peptides and other large biological molecules. Mass spectrometry (MS) is a useful tool for determining the identity of molecules based on their interaction with electromagnetic fields. If coupled with another method like infrared (IR) vibrational spectroscopy, MS can provide structural information, but in its own right, MS can only provide the mass-to-charge (m/z) ratio of the fragments produced, which may not be enough information to determine the mechanism of the collision-induced dissociation (CID) of the molecule. In this case, theoretical calculations provide a useful companion for MS data and yield clues about the energetics of the dissociation. In this study, negative ion electrospray tandem MS was used to study the CID of the deprotonated dipeptide glycine-serine (Gly-Ser). Though negative ion MS is not as popular a choice as positive ion MS, studies by Bowie et al. show that it yields unique clues about molecular structure which complement positive ion spectroscopy, such as characteristic fragmentations like the loss of formaldehyde from the serine residue.2 The increase in the collision energy in the mass spectrometer alters the flexibility of the dipeptide backbone, enabling isomerizations (reactions not resulting in a fragment loss) and dissociations to take place. The mechanism of the CID of Gly-Ser was studied using two computational methods, B3LYP/6-311+G* and M06-2X/6-311++G**. The main pathway for molecular dissociation was analyzed in 5 conformers in an attempt to verify the initial mechanism proposed by Dr. James Swan after examination of the MS data. The results suggest that the loss of formaldehyde from serine, which Bowie et al. indicates is a characteristic of the presence of serine in a protein residue, is an endothermic reaction that is made possible by the conversion of the translational energy of the ion into internal energy as the ion collides with the inert collision gas. It has also been determined that the M06-2X functional¿s improved description of medium and long-range correlation makes it more effective than the B3LYP functional at finding elusive transition states. M06-2X also more accurately predicts the energy of those transition states than does B3LYP. A second CID mechanism, which passes through intermediates with the same m/z ratio as the main pathway for molecular dissociation, but different structures, including a diketopiperazine intermediate, was also studied. This pathway for molecular dissociation was analyzed with 3 conformers and the M06-2X functional, due to its previously determined effectiveness. The results suggest that the latter pathway, which meets the same intermediate masses as the first mechanism, is lower in overall energy and therefore a more likely pathway of dissociation than the first mechanism.