This project applies structure-activity relationship (SAR), structure-based and database mining approaches to study ligand-protein interactions. To support these studies, we have developed a relational database system called EDinburgh University Ligand Selection System (EDULISS 2.0) which stores the structure-data files of +5.5 million commercially available small molecules (+4.0 million are recognised as unique) and over 1,500 various calculated molecular properties (descriptors) for each compound. A user-friendly web-based interface for EDULISS 2.0 has been established and is available at http://eduliss.bch.ed.ac.uk/. We have utilised PubChem bioassay data from an NMR based screen assay for a human FKBP12 protein (PubChem AID: 608). A prediction model using a Logistic Regression approach was constructed to relate the assay result with a series of molecular descriptors. The model reveals 38 descriptors which are found to be good predictors. These are mainly 3D-based descriptors, however, the presence of some predictive functional groups is also found to give a positive contribution to the binding interaction. The application of a neural network technique called Self Organising Maps (SOMs) succeeded in visualising the similarity of the PubChem compounds based on the 38 descriptors and clustering the 36 % of active compounds (16 out of 44) in a cluster and discriminating them from 95 % of inactive compounds. We have developed a molecular descriptor called the Atomic Characteristic Distance (ACD) to profile the distribution of specified atom types in a compound. ACD has been implemented as a pharmacophore searching tool within EDULISS 2.0. A structure-based screen succeeded in finding inhibitors for pyruvate kinase and the ligand-protein complexes have been successfully crystallised. This study also discusses the interaction of metal-binding sites in metalloproteins. We developed a database system and web-based interface to store and apply geometrical information of these metal sites. The programme is called MEtal Sites in Proteins at Edinburgh UniverSity (MESPEUS; http://eduliss.bch.ed.ac.uk/MESPEUS/). MESPEUS is an exceptionally versatile tool for the collation and abstraction of data on a wide range of structural questions. As an example we carried out a survey using this database indicating that the most common protein types which contain Mg-OATP-phosphate site are transferases and the most common pattern is linkage through the β- and γ-phosphate groups.

Transient change of cytosolic calcium level leads to physiological actions, which are modulated by the intracellular calcium stores, and gated by membrane calcium channels/pumps. To closely monitor calcium dynamics there is a pressing need to develop calcium sensors that are targeted to high calcium environment such as the ER/SR with relatively low binding affinity and fast kinetic properties to complement the current calcium indicator toolkits. In this dissertation, the development of fast red florescent calcium binding protein using the protein design is reported. The results show the calcium dependent fluorescence increase of mCherry mutant MCD1 (RapidER) and MCD15 (RapidER’) is able to monitor the ER calcium release in several cell lines responding to perturbations of extracellular calcium signaling. The specific targeting to the ER membrane was achieved by fusing the ryanodine receptor 1 transmembrane domains for the spatio-temporal calcium imaging. To understand the underlying mechanism of calcium binding induced fluorescence increase in the designed calcium sensor CatchER, the fluorescence lifetime of CatchER was determined in calcium free and bound forms using time resolved florescence spectroscopy. The results suggest that calcium binding inhibits the geminate quenching, resulting in a longer lifetime when the anionic form is indirectly excited at 395 nm. It is believed that such unique calcium-induced lifetime change can be applied to monitor calcium signaling in cell imaging. NMR spectroscopy was used to investigate the protein-protein/ligand interaction in this dissertation. The residual dipolar coupling and T1, T2, NOE dynamic study were carried out to understand the binding mode of CaM and the N-terminal intracellular loop of connexin 43. The results show that both N and C terminal domains of Ca2+-CaM contact with the peptide, leading to a partially unwound and bending central helix of CaM. The ligand binding induced conformational change was demonstrated by selectively labeled proteins including extracellular domain of calcium sensing receptor and the bacterial membrane protein SecA fragments C34 and N68.