Molecular dynamics (MD) is a powerful tool that can be applied to protein folding and protein structure. MD allows for the calculation of movement, and final position, of atoms in a biomolecule. These movements can be used to investigate the pathways that allow proteins to fold into energetically favorable structures. While MD is very useful, it still has its limitations. Most notable, computing power and time are of constant concern. Protein structure is inherently important due to the direct link between the structure of a protein and its function. One of the four levels of protein structure, the secondary structure, is the first level to accommodate for the three-dimensional shape of a protein. The main driving force behind secondary structure is hydrogen bonding, which occurs between the carboxyl oxygen and the amine hydrogen of the backbone of a peptide. Determining a greater link between hydrogen bond patterns and types of secondary structure can provide more insight on how proteins fold. Because molecular dynamics allows for an atomic level view of the dynamics behind protein folding/unfolding, it becomes very useful in observing the effects of particular hydrogen bond patterns on the folding pathway and final structure formed of a protein. Using molecular dynamic simulations, a series of experiments in an attempt to alter structure, hydrogen bonding, and folding patterns, can be performed. This information can be used to better understand the driving force of secondary structure, and use the knowledge gained to manipulate these simulations to force folding events, and with that, desired secondary structure features.

Peptide tags are ubiquitous in the life sciences, with roles including purification and selective labeling of proteins. Because peptide tags are small they have a limited surface area for binding and hence usually form low affinity protein interactions. These weak interactions limit the uses of peptide tags in cases that require resistance to forces generated with macromolecular architectures or protein motors. Hence a way to create a covalent interaction with a peptide tag would be useful. It was found possible to create a covalent bond-forming peptide tag using the spontaneous isopeptide chemistry of the CnaB2 domain from the Gram-positive bacterium Streptococcus pyogenes. In the CnaB2 domain a reactive Lysine forms an isopeptide bond with an Aspartic acid, catalyzed by a Glutamic acid, creating an internal covalent linkage. Subsequently it was shown that the CnaB2 domain could be split into two parts, a domain with the Lysine and Glutamic acid called SpyCatcher and a peptide with the Aspartic acid called SpyTag, such that the isopeptide covalent linkage can be formed when SpyCatcher/SpyTag are mixed together. SpyCatcher/SpyTag was applied in this thesis and showed functionality in a wide array of scenarios. SpyCatcher/SpyTag covalently linked within the cytosol of E. coli, on surface membrane proteins of HeLa cells, and regardless of whether SpyTag was located on the N- or C-terminus or an internal site. Crystal structures of SpyCatcher/SpyTag were then obtained and it was found possible to shrink the SpyCatcher by 32 residues to a core domain of 83 residues. To create an even smaller covalent linkage system, SpyCatcher was split further to generate a protein (SpyLigase) ligating two peptide tags. The β-sheet with the reactive Lysine was removed from SpyCatcher and called KTag. SpyLigase could covalently link SpyTag and KTag. SpyLigase-induced ligation was independent of the location of SpyTag/KTag on the target proteins and was applied to create affibody polymers, which were shown to improve magnetic isolation of cells with low tumor antigen expression. Through this work protein-protein covalent linkage systems were refined and generated that have future applications for the creation of unique macromolecular structures, cellular labeling, and protein cyclization.

Protein tertiary structure is more conserved than amino acid sequence, leading to a diverse range of functions observed in the same fold. Despite < 20 % overall sequence identity, cytochromes P450 all have the same fold. Bacterial Class I P450s receive electrons from a highly specific, often unidentified, ferredoxin, in which case the hemoprotein is termed “orphaned”. CYP199A2, a Class I P450, accepts electrons from ferredoxins Pux and HaPux. Five orientation-dependent and one orientation-independent DEER measurements on paramagnetic HaPux and spin-labelled CYP199A2 yielded vector restraints, which were applied to building a model of the CYP199A2:HaPux complex in silico. A different binding mode was observed compared to P450cam:Pdx and P450scc:Adx, both recently elucidated by X-ray crystallography. This protocol was also applied to the CYP101D1:Arx complex. The first three measurements indicate that this heterodimer does not have a similar orientation to CYP199A2:HaPux, P450cam:Pdx, or P450scc:Adx. P450cam was fused to putidatredoxin reductase (PdR) to explore the kinetic effects with a view to improving electron transfer to orphan P450s. Heme incorporation of this enzyme depends on linker length. In whole cells, the fusion was more active after longer incubations. In vitro kinetics of the fusion exhibited some co-operativity and enhanced kinetics over the unfused system under steady-state conditions. The putative iron-sulfur biosynthesis ferredoxin PuxB had been engineered by rational mutagenesis to support catalysis by CYP199A2. It was confirmed this arose from improved protein-protein recognition. Engineering of E. coli ferredoxin based on these findings was carried out, resulting in electron-transfer to CYP199A4 from a novel engineered alien ferredoxin.

Oxygen dependent prolyl-4-hydroxylation of the alpha-subunit of the hypoxia inducible transcription factor (HIF-alpha) plays an essential role in the hypoxic response. Hydroxylation of proline residues in the N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the affinity of HIF-alpha to the von Hippel-Lindau protein (pVHL) by approx. 1000 fold so signalling for HIF-alpha degradation. With limiting oxygen, HIF-alpha hydroxylation slows, it dimerises with HIF-beta and activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilises the triple helix structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Crystal structures of PHD2 in complex with CODD were determined in the current study. Together with biochemical analyses, the results demonstrate that catalysis involves a mobile region of PHD2 that encloses the hydroxylation site and stabilises the PHD2.Fe(II).2OG complex. When bound to PHD2 the pyrrolidine ring of the non-hydroxylated proline-residue adopts a C⁴-endo conformation. Evidence is provided that 4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to the C⁴-exo state, as observed when hydroxylated HIF-alpha is bound to pVHL and in collagen. The results help to rationalise NODD/CODD selectivity data for PHD isoforms and the effects of clinically observed mutations on PHD2 catalysis. Analyses on the interaction of nitric oxide with PHD2 are described and discussed with respect to regulation of the hypoxic response by nitric oxide.

In order to associate with the cytoplasmic leaflet of the plasma membrane, many cytosolic signalling proteins possess a distinct lipid binding domain as part of their overall fold. Here, a multiscale simulation approach has been used to investigate three membrane-binding proteins involved in cellular processes such as growth and proliferation. The pleckstrin homology (PH) domain from the general receptor for phosphoinositides 1 (GRP1-PH) binds phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) with high affinity and specificity. To investigate how this peripheral protein is able to locate its target lipid in the complex membrane environment, Brownian dynamics (BD) simulations were employed to explore association pathways for GRP1-PH binding to PI(3,4,5)P₃ embedded in membranes with different surface charge densities and distributions. The results indicated that non-PI(3,4,5)P₃ lipids can act as decoys to disrupt PI(3,4,5)P₃ binding, but that at approximately physiological anionic lipid concentrations steering towards PI(3,4,5)P₃ is actually enhanced. Atomistic molecular dynamics (MD) simulations revealed substantial membrane penetration of membrane-bound GRP1-PH, evident when non-equilibrium, steered MD simulations were used to forcibly dissociate the protein from the membrane surface. Atomistic and coarse grained (CG) MD simulations of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumour suppressor, which also binds PI(3,4,5)P₃, detected numerous non-specific protein-lipid contacts and anionic lipid clustering around PTEN that can be modulated by selective in silico mutagenesis. These results suggested a dual recognition model of membrane binding, with non-specific membrane interactions complementing the protein-ligand interaction. Molecular docking and MD simulations were used to characterise the lipid binding properties of kindlin-1 PH. Simulations demonstrated that a dynamic salt bridge was responsible for controlling the accessibility of the binding site. Electrostatics calculations applied to a variety of PH domains suggested that their molecular dipole moments are typically aligned with their ligand binding sites, which has implications for steering and ligand electrostatic funnelling.

Careful treatment of water molecules in ligand-protein interactions is required in many cases if the correct binding pose is to be identified for molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors (iGluRs), a family of ligand gated ion channels that are responsible for a majority of the fast synaptic neurotransmission in the central nervous system that are thought to be essential in memory and learning. Thus, pharmacological intervention at these neuronal receptors is a valuable therapeutic strategy. This thesis relies on various computational studies and X-ray crystallography to investigate the role of ligand-water mediated interactions in iGluRs bound to glutamate and α-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA). Comparative molecular dynamics (MD) simulations of each subtype of iGluRs bound to glutamate revealed that crystal water positions were reproduced and that all but one water molecule, W5, in the binding site can be rearranged or replaced with water molecules from the bulk. Further density functional theory calculations (DFT) have been used to confirm the MD results and characterize the energetics of W5 and another water molecule implicated in influencing the dynamics of a proposed switch in these receptors. Additional comparative studies on the AMPA subtypes of iGluRs show that each step of the calculation must be considered carefully if the results are to be meaningful. Crystal structures of two ligands, glutamate and AMPA revealed two distinct modes of binding when bound to an AMPA subtype of iGluRs, GluA2. The difference is related to the position of water molecules within the binding pocket. DFT calculations investigated the interaction energies and polarisation effects resulting in a prediction of the correct binding mode for glutamate. For AMPA alternative modes of binding have similar interaction energies as a result of a higher internal energy than glutamate. A combined MD and X-ray crystallographic study investigated the binding of the ligand AMPA in the AMPA receptor subtypes. Analysis of the binding pocket show that AMPA is not preserved in the crystal bound mode and can instead adopt an alternative mode of binding. This involves a displacement of a key water molecule followed by AMPA adopting the pose seen by glutamate. Thus, this thesis makes use of various studies to assess the energetics and dynamics of water molecules in iGluRs. The resulting data provides additional information on the importance of water molecules in mediating ligand interactions as well as identifying key water molecules that can be useful in the de novo design of new selective drugs against iGluRs.

Computational methods, including homology modelling, in-silico dockings, and molecular dynamics simulations have been used to study the functional dynamics and interactions of K⁺ channels. Molecular models were built of the inwardly rectifying K⁺ channel Kir2.2, the bacterial homolog K⁺ channel KirBac3.1, and the twin pore (K2P) K⁺ channels TREK-1 and TRESK. To investigate the electrostatic energy profile of K⁺ permeating through these homology models, continuum electrostatic calculations were performed. The primary mechanism of KirBac3.1 gating is believed to involve an opening at the helix bundle crossing (HBC). However, simulations of Kir channels have not yet revealed opening at the HBC. Here, in simulations of the new KirBac3.1-S129R X-ray crystal structure, in which the HBC was trapped open by the S129R mutation in the inner pore-lining helix (TM2), the HBC was found to exhibit considerable mobility. In a simulation of the new KirBac3.1-S129R-S205L double mutant structure, if the S129R and the S205L mutations were converted back to the wild-type serine, the HBC would close faster than in the simulations of the KirBac3.1-S129R single mutant structure. The double mutant structure KirBac3.1-S129R-S205L therefore likely represents a higher-energy state than the single mutant KirBac3.1-S129R structure, and these simulations indicate a staged pathway of gating in KirBac channels. Molecular modelling and MD simulations of the Kir2.2 channel structure demonstrated that the HBC would tend to open if the C-linker between the transmembrane and cytoplasmic domain was modelled helical. The electrostatic energy barrier for K⁺ permeation at the helix bundle crossing was found to be sensitive to subtle structural changes in the C-linker. Charge neutralization or charge reversal of the PIP2-binding residue R186 on the C-linker decreased the electrostatic barrier for K⁺ permeation through the HBC, suggesting an electrostatic contribution to the PIP2-dependent gating mechanism. Multi-scale simulations determined the PIP2 binding site in Kir2.2, in good agreement with crystallographic predictions. A TREK-1 homology model was built, based on the TRAAK structure. Two PIP2 binding sites were found in this TREK-1 model, at the C-terminal end, in line with existing functional data, and between transmembrane helices TM2 and TM3. The TM2-TM3 site is in reasonably good agreement with electron density attributed to an acyl tail in a recently deposited TREK-2 structure.

Membrane proteins represent about one third of all known vertebrate proteins and over half of the current drug targets. Knowledge of their three-dimensional (3D) structure is worth millions of pounds to the pharmaceutical industry. Yet experimental structure elucidation of membrane proteins is a slow and expensive process. In the absence of experimental data, computational modelling tools can be used to close the gap between the numbers of known protein sequences and structures. However, currently available structure prediction tools were developed with globular soluble proteins in mind and perform poorly on membrane proteins. This thesis describes the development of a modelling approach able to predict accurately the structure of transmembrane domains of proteins. In this thesis we build a template-based modelling framework especially for membrane proteins, which uses membrane protein-specific information to inform the modelling process.Firstly, we develop a tool to accurately determine a given membrane protein structure's orientation within the membrane. We offer an analysis of the preferred substitution patterns within the membrane, as opposed to non-membrane environments, and how these differences influence the structures observed. This information is then used to build a set of tools that produce better sequence alignments of membrane proteins, compared to previously available methods, as well as more accurate predictions of their 3D structures. Each chapter describes one new piece of software or information and uses the tools and knowledge described in previous chapters to build up to a complete accurate model of a transmembrane domain.

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 &Aring; resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

Ionenkanäle spielen eine wichtige Rolle in vielen physiologischen Prozessen. Während manche Kanäle hochspezifisch für eine Ionensorte sind, sind andere Kanäle weniger selektiv. Tetramere Ionenkanäle weisen eine gemeinsame Grundarchitektur auf, von der nur ionotrope Glutamatrezeptoren (iGluRs) abweichen, deren Struktur im Vergleich zu den anderen invertiert ist. Eine Untergruppe der iGluRs stellen α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Rezeptoren (AMPARs) dar, welche im Zentralnervensystem von Wirbeltieren die Mehrheit der exzitatorischen Neurotransmission übernehmen. Eine einzelne posttranskriptionelle Veränderung (Glutamin zu Arginin) an der Spitze des Selektivitätsfilters (SF, Q/R Stelle) von AMPAR-Typ 2 (GluA2) macht den Kanal quasi undurchlässig für Ca2+. Das Ziel dieser Arbeit war das Erstellen einer GluA2-Kanalchimäre mit Hilfe des bakteriellen Kanals NaK. Mutation rund um die Q/R Stelle wurden nicht toleriert von der Chimäre, während C-terminale Mutationen des SF stabil waren und für strukturelle Studien verwendet wurden. Die Entfernung einer Aminosäure im Vergleich zum NaK Wildtyp erzeugte eine Begradigung des SF und eine Erweiterung der wassergefüllten Ausbuchtung. Überraschenderweise kristallisierten die NaK Chimären überwiegend in zweifach symmetrischer Anordnung. Vor allem im SF kam es zu ausgeprägter lokaler Asymmetrie, unabhängig von der Ionenzusammensetzung. Um die Flexibilität des SF näher zu untersuchen, wurden Aminosäuren von NaK in GluA2 integriert und mithilfe von Patch-clamp Elektrophysiologie untersucht. Gleichsam wie in NaK, wurden Mutationen an der Filterspitze nicht toleriert, wohingegen die C-terminale Hälfte problemlos ausgetauscht werden konnte. Die funktionelle Integrität der NaK Chimäre wurde mithilfe von Einzelkanalmessungen in Bilayern überprüft. Zusätzlich wurden, auf Basis der gelösten Strukturen, Molekulardynamiksimulationen durchgeführt, welche einen dynamischen Einblick in den Permeationsmechanismus erlauben. / Ion channels play an important role in many physiological processes, for example the generation of action potentials. While some channels display high selectivity for one ion species, others are more promiscuous. All tetrameric cation channels share the same principal architecture, but the transmembrane domain of ionotropic glutamate receptors (iGluRs) is inverted relative to the other members. A subfamily of iGluRs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), mediates the vast majority of excitatory neurotransmission in the vertebrate central nervous system. In AMPA-type 2 iGluRs (GluA2), a single post-transcriptional modification (glutamine to arginine) at the tip of the selectivity filter (SF, Q/R site) renders the channel Ca2+ impermeable. The aim of this thesis was therefore to create an AMPAR pore mimic by using the bacterial channel NaK. Unfortunately, mutations around the Q/R site abolished expression of the NaK-GluA2 chimera, while C-terminal mutations of the SF were stable and could be used for structural studies. The shortening of the SF by one amino acid caused a straightening and opened an extended water-filled vestibule. Strikingly, most of the tested chimeras exhibited twofold symmetry with strong local asymmetry in the mutated part of the SF independent of the ion type present. For functional tests, residues from NaK wt were also swapped into GluA2. N-terminal mutations abolished the current response, whereas C-terminal mutations behaved wt-like. Single-channel bilayer experiments confirmed the functional integrity of the NaK-GluA2 chimera. Additionally, extensive molecular dynamics simulations, based on the solved structures, were carried out alongside. In summary, it could be demonstrated that the SF of the non-selective NaK-GluA2 chimera is highly flexible and accommodated all tested ions. Ion binding is accompanied by local asymmetric rearrangements, possibly creating an energetically simple way to allow permeation.