Investigation of Protein Structure and Dynamics

Abstract

This thesis addresses three aspects of protein function:Part I describes the structural investigation of a calmodulin-peptide complex by NMR spectroscopy. The intracellular calcium sensor calmodulin, model system for a two domain protein, is known to interact with a large variety of different peptides, among them the IQ-motif in the cytoplasmic C-terminal tail of the CaV α1 subunit of voltage gated ion channels. A crystal structure of the CaM/IQ-motif complex showed that the CaM N-terminal lobe adopts two conformations suggesting internal conformational plasticity. In order to explore this plasticity and to work towards motional models of the underlying processes, paramagnetic alignment was employed to probe domain dynamics using residual dipolar couplings (RDC), a versatile NMR parameter. The results are compared to existing X-ray structures of the complex. It is concluded that in solution a larger conformational space is sampled by calmodulin than predicted by the available structures, but that these structures are likely to be a subset of this conformational space.Part II describes the development of a web based tool that can aid with the identification of protein-protein cross-links and peptide-based post-translational modifications by mass spectrometry. Although several well-established and powerful software suites are available (e.g. Mascot [98], Sequest [99]), these are not suitable for addressing a number of scenarios opened up by recent developments. This is especially true in the case of large peptide modifications. Such modifications include post-translational modifications (PTM), most notably Ubiquitin (Ubq) and the Ubiquitin-like modifier (Ubl) families, but also general protein-protein cross-linking. The latter may be endogenous - i.e. disulphide bridges - or engineered through the use of cross-linking reagents, to study the three-dimensional structure of, or the interactions between proteins. To this end, a large number of specialised programmes addressing specific questions have been proposed, each with individual advantages and disadvantages. In contrast, the ChopTools suite is not limited to a specific modifier or affected by the drawbacks of predictive approaches. Instead, it provides a convenient and ubiquitous way of reformulating the question through the generation of fragment library files. This in turn allows well-established programmes to address the questions at hand thereby bridging the gap between existing analysis schemes and new experimental setups.Part III describes progress towards determining a high resolution structure of the Dead end protein. This protein has been shown to play a crucial role in fate maintenance, survival and migration of primordial germ cells in the model organism zebrafish (Danio rerio). In addition, a particular mutation in the gene encoding the mouse Dead end orthologue results in germ cell loss and testicular tumours. Close homologues of the protein are also found in african clawed frog, chick and human, suggesting a central and general role for this protein in germ cell development. Primary sequence analysis and functional characterisation suggest that the essential core region of the protein consists of a putative RNA or protein interaction domain with an RNP fold and another domain of similar size with unknown structure. Recent studies have shown that Dead end is capable of protecting mRNAs from microRNA (miRNA) mediated repression. A wide range of protein expression and purification strategies are discussed in the context of the Dead end protein. Although no sample amen! able to structural investigation could be obtained, the work has laid the foundation of future biophysical and biochemical investigations of this disease relevant protein, and has established a protocol for the preparation of dilute recombinant protein solutions that may prove valuable in the generation of antibodies to the Dead end protein.