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Abstract: Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes residual electron and anomalous density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data-collection and analysis strategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly disordered sections of crystal structures.

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Abstract: In the course of this work, structural and biochemical information about a transcription factor from the pathogenic Mycobacterium avium ssp. paratuberculosis and of an L-RNA aptamer, in complex with the orexigenic Peptide ghrelin, was obtained. In the first part of the thesis the peroxide sensitive, transcription factor Ferric Uptake Regulator (FurA) from Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent for paratuberculosis in ruminants and especially in livestock, was studied. This disease is often fatal and responsible for huge economic losses for the livestock industry worldwide. Here, first structural information about MAP FurA is presented. For the first time, the open and the closed confirmation of MAP FurA were observed, where the open form is putatively able to bind DNA to regulate gene expression. The open form is the metalized, unoxidized MAP FurA. It was shown, that MAP FurA is able to bind manganese and iron, as well as zinc. This might indicate, that upon binding of different metal ions, a fine-tuning mechanism of regulation is possible, depending on the current cytosolic composition. The apo-protein and the oxidized form are in the closed form, indicating that these forms are not able to bind DNA. When peroxide stress is occurring, the iron gets oxidized and dissociated from the protein resulting in a conformational change. Crystallization trials were performed and first initial microcrystals were obtained. Further optimization is needed to obtain structural information about the metal binding and DNA interaction. The second part of this thesis provides structural insights into the Ghrelin•NOX-B11 complex and presents several methods that can be used for phase retrieval for nucleic Acid containing crystals. In the 1990s the development of SELEX (Systematic Evolution of Ligands by Exponential enrichment) led to the development of aptamers, a novel group of DNA or RNA molecules that bind to their cognate targets with high affinity via their 3D structure. Spiegelmers were developed by the NOXXON Pharma AG and are a Special class of aptamers that consist of unnatural L-nucleotides and are hence not susceptible to endo- and exo-nucleases. NOX-B11 is a Spiegelmer that binds the active octanoyl-ghrelin with high affinity and effectiveness has been demonstrated in vitro and in vivo. Ghrelin is an orexigenic peptide that is associated with various physiological processes, mostly in the regulation of food metabolism and hunger. It is therefore an important drug target. The active form of ghrelin, which binds its receptor (growth hormone secretagogue receptor (GHSR)1a), has a posttranslational, and for mammals unique, modification, namely a fatty acid side chain bound at Ser3. Here, first structural information About complex formation was obtained. It was shown, that the L-RNA and the complex are highly stable. In situ DLS studies revealed a conformational change of the Spiegelmer upon ghrelin addition and stability of the L-RNA and the complex was monitored over 40 days. Furthermore, SAXS measurements demonstrated the conformational change as well, as the L-RNA transitions from an elongated molecule to a more compact complex with ghrelin. Extensive crystallization trials were performed and it was possible to obtain X-ray diffraction suitable crystals. X-ray diffraction data were collected from a single crystal to a resolution of 2.65 Å and the space group was determined to be C2. Since no structural information about the Spiegelmer, nor ghrelin, was known, several Phase retrieval methods were applied. The phase problem of crystallography is still the main bottleneck of structure determination, as only the amplitudes of a reflection can be measured and the phases are not observed. Native SAD is a comparatively new Approach for phase retrieval, using the intrinsic atoms (sulfur for proteins, phosphorous for nucleic acids) as anomalous scattering atoms.

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Abstract: The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity.

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Abstract: A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.

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Abstract: Calbindin-D28K is a widely expressed calcium-buffering cytoplasmic protein that is involved in many physiological processes. It has been shown to interact with other proteins, suggesting a role as a calcium sensor. Many of the targets of calbindin-D28K are of therapeutic interest: for example, inositol monophos­phatase, the putative target of lithium therapy in bipolar disorder. Presented here is the first crystal structure of human calbindin-D28K. There are significant deviations in the tertiary structure when compared with the NMR structure of rat calbindin-D28K (PDB entry 2g9b), despite 98% sequence identity. Small-angle X-ray scattering (SAXS) indicates that the crystal structure better predicts the properties of calbindin-D28K in solution compared with the NMR structure. Here, the first direct visualization of the calcium-binding properties of calbindin-D28K is presented. Four of the six EF-hands that make up the secondary structure of the protein contain a calcium-binding site. Two distinct conformations of the N-terminal EF-hand calcium-binding site were identified using long-wavelength calcium single-wavelength anomalous dispersion (SAD). This flexible region has previously been recognized as a protein–protein interaction interface. SAXS data collected in both the presence and absence of calcium indicate that there are no large structural differences in the globular structure of calbindin-D28K between the calcium-loaded and unloaded proteins.