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Abstract: The functioning of almost all biological macromolecules depends on their ability to form specific interactions with one or more other species, such as other macromolecules or small molecule ligands, and quantitative characterization of these interactions often leads to a greatly improved understanding of the role of the particular protein or nucleic acid in its cellular context. All common biological macromolecules exhibit the phenomenon of circular dichroism (CD), the differential absorption of left-and right-handed circularly polarized light, and this CD signal can be exquisitely sensitive to the conformational state of the molecule. The fact that complex formation is frequently accompanied by conformational changes means that CD spectroscopy is one of the few techniques that allows one to address simultaneously both the structural and the energetic aspects of these interactions. Because each experimental system is different, and will inevitably present its own particular set of challenges, the process of determining an equilibrium dissociation constant cannot be presented as a single simple protocol that can be applied in every situation. In this chapter we therefore provide a general overview of CD-based approaches with emphasis on experimental design, data collection, and data analysis. We describe the methods that may be used when a suitable signal change accompanies formation of the complex as well as those that may sometimes be used when there is no signal change.

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Abstract: An FePd thin film sample, showing magnetic stripe domains as imaged by magnetic force microscopy, has been measured by soft X-ray resonant magnetic scattering in reflection geometry. Illumination with coherent radiation, produced by inserting a 20 mum pinhole in front of the sample, leads to a magnetic speckle pattern in the scattered intensity that gives access to the domain morphology. Application of an in-plane magnetic field for a few seconds gives a strong change in the observed intensity fluctuations, which indicates a large degree of variation between the two patterns taken before and after field exposure. From the speckle pattern we calculate a degree of coherence of beta = 0.5 for the incident beam.

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Abstract: The recognition of the correct substrate and rejection of the incorrect substrate by RNA-modifying enzymes is of great importance to the process of posttranscriptional base modification. Between 2001 and 2008, structures of nine different rRNA and tRNA base-modifying enzymes in complex with RNA were published and these revealed a plethora of mechanisms for substrate recognition. While some complexes appear to be the result of the RNA being bound primarily as a rigid body, a significant number of enzymes remodel the RNA upon binding, either to increase the affinity of the interaction and/or access an otherwise buried base. Pertinent examples of remodelled RNA are observed in the structures of the ArcTGT-tRNA and RlmD-rRNA complexes. In a number of RNA-modifying enzymes it is clear that there is a degree of modularity, such that the substrate specificity is partly defined by a domain(s) that recognises a particular feature in the RNA while the modification is performed by a suitably positioned catalytic domain. With this chapter we will first review the general principles of protein-RNA interactions and modularity in RNA-modifying enzymes before looking at how they are combined to define substrate specificity in a number of interesting protein-RNA complexes.

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Abstract: MTH909 is the Methanothermobacter thermautotrophicus orthologue of Saccharomyces cerevisiae TAN1, which is required for N4-acetylcytidine formation in tRNA. The protein consists of an N-terminal near-ferredoxin-like domain and a C-terminal THUMP domain. Unlike most other proteins containing the THUMP domain, TAN1 lacks any catalytic domains and has been proposed to form a complex with a catalytic protein that is capable of making base modifications. MTH909 has been cloned, overexpressed and purified. The molecule exists as a monomer in solution. X-ray data were collected to 2.85 Å resolution from a native crystal belonging to space group P6122 (or P6522), with unit-cell parameters a = 69.9, c = 408.5 Å.