Algorithms for automated assignment of solution-state and solid-state protein NMR spectra.

Protein nuclear magnetic resonance spectroscopy (Protein NMR) is an invaluable analytical technique for studying protein structure, function, and dynamics. There are two major types of NMR spectroscopy that are used for investigation of protein structure – solution-state and solid-state NMR. Solution-based NMR spectroscopy is typically applied to proteins of small and medium size that are soluble in water. Solid-state NMR spectroscopy is amenable for proteins that are insoluble in water. In the vast majority NMR-based protein studies, the first step after experiment optimization is the assignment of protein resonances via the association of chemical shift values to specific atoms in a protein macromolecule. Depending on the quality of the spectra, a manual protein resonance assignment process often requires a considerable amount of time, from weeks to months-worth of effort even, by an experienced NMR spectroscopist . The resonance assignment processes for solution-state and solid-state protein NMR studies are conceptually similar, but have distinct differences due to the utilization of different NMR experiments and to the use of different resonances for grouping peaks into spin systems. Currently, there is a shortage of robust, effective software tools that can perform solid-state protein resonance assignment and there is no general software that can perform both solution-state and solid-state protein resonance assignment in a reliable, automated fashion. Hence, the motivation of this research is to design and implement algorithms and software tools that will automate the resonance assignment problem. As a result of this research, several algorithms and software packages that aid several important steps in the protein resonance assignment process were developed. For example, the nmrstarlib software package can access and utilize data deposited in the NMR-STAR format; the core of this library is the lexical analyzer for NMR-STAR syntax that acts as a generator-based state-machine for token processing. The jpredapi software package provides an easy-to-use API to submit and retrieve results from secondary structure prediction server. The single peak list and pairwise peak list registration algorithms address the problem of multiple sources of variance within single peak list and between different peak lists and is capable of calculating the match tolerance values necessary for spin system grouping. The single peak list and pairwise peak list grouping algorithms are based on the well-known DBSCAN clustering algorithm and are designed to group peaks into spin systems within single peak list as well as between different peak lists

Development of NMR methods for the structural elucidation of large proteins

Ph.DDOCTOR OF PHILOSOPH

Insights into the structures and dynamics of the pathogen secreted effectors AVR3A11 and tarp through the application of NMR spectroscopy

The lifecycles of obligate pathogenic and parasitic microorganisms depend on a myriad of interactions with their hosts at the molecular level. The class of bacterial proteins directly responsible for these inter-organism interactions have been termed e�ector proteins and can function in either an extracellular or, once secreted into the host cell, an intracellular environment. Primarily through the use of nuclear magnetic resonance spectroscopy (NMR), I have investigated the biophysical properties of two such bacterial e�ector proteins. TARP (translocated actin recruiting protein) is a largely disordered 100 kDa e�ector, common to all chlamydial species, which functions to remodel the host actin cytoskeleton to facilitate the internalisation of the chlamydial cell. Using constructs of TARP comprising an expected actin binding domain, I have shown through NMR chemical shift indexing and 15N relaxation that although the unbound domain is intrinsically disordered a short region, which aligns to other helical actin binding domains, maintains some helical propensity. Furthermore, these residues map to chemical shift variations in the bound state and the Kd for the interaction has also been determined using isothermal titration calorimetry. AVR3a11 is an 8 kDa e�ector from the pepper pathogen Phytophthora capsici that has been shown to inhibit plant programmed cell death. Using a combination of 2D and 3D NMR experiments I have assigned the majority of the backbone and side-chain resonances from the structured regions of AVR3a11. Through the acquisition and analysis of 13C and 15N edited HSQCNOESY spectra I have also calculated a water re�ned, structural ensemble for AVR3a11. Additionally, analysis of the slow (H:D exchange) and fast (T1, T2 and heteronuclear NOE) dynamic regimes, describes AVR3a11 as a relatively tightly folded helical bundle which also exhibits a signi�cant degree of conformational exchange

Cloning, expression and characterisation of the starter module from indanomycin biosynthesis

Nonribosomal peptides and polyketides form important classes of pharmaceutical agents. Several architectures of biosynthetic machinery exist for the construction of these structurally diverse molecules. Many attempts to engineer these proteins have concentrated on the multimodular type I polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). Here, work was carried out to express the NRPS module responsible for starter unit formation from indanomycin biosynthesis in E. coli. Three synthetic genes, for IdmI, IdmJ and IdmK, were cloned and subsequently used in expression trials. Despite multiple attempts, IdmI was always expressed as an insoluble protein. IdmJ was expressed as a soluble protein, and an unexpected post-translational modification was found and investigated. Mutagenesis studies suggested that the unknown post-translational modification was occurring at a cysteine 127. IdmK, the carrier protein, was expressed in a soluble form with good yield. Analysis by mass spectrometry showed that, surprisingly, E. coli was able to phosphopantetheinylate IdmK, which is required for a functional module. Preliminary structure determination was carried out by X-ray crystallography. A complete 3D structure was obtained using NMR spectroscopy of the [15N] and [13C, 15N] labelled protein. Structure determination was performed using CS-ROSETTA, which only uses chemical shift data, and ARIA, which assigns ambiguous NOE distance restraints. Both structure calculations produced comparable structures for IdmK. The structure showed a 3 α-helix bundle, with the same topology, lengths and locations of helices as other carrier proteins. Initial investigations into holo-IdmK suggest that the phosphopantetheine co-factor is directed into the hydrophobic core of the protein. This research has set up the system for future studies to engineer the pathway for novel product biosynthesis