The bacteria Streptococcus pyogenes produces a multitude of proteins which interact with and alter the functions of the host immune system. Two such proteins, Endoglycosidase S (EndoS) and Immunoglobulin G-degrading enzyme from S. pyogenes (IdeS) are able to specifically alter the effector functions of immunoglobulin G (IgG). EndoS is a glycoside hydrolase which removes the conserved <i>N</i>-linked glycan from IgG Fc whereas IdeS is a cysteine protease that cleaves the exible protein hinge of IgG. The activity of both proteins results in the reduced ability of IgG to elicit immune responses through Fc receptor binding and complement activation. Amongst other applications, both EndoS and IdeS are actively being explored as new therapeutics for IgG-mediated autoimmune diseases. Given the therapeutic potential of EndoS and IdeS, experiments were designed to investigate the structural and functional characteristics of these enzymes in an effort to understand their specficity for and activity against IgG. Here, bioinformatic and biophysical characterisation of EndoS identified subdomains outside of the catalytic domain which contribute to glycoside hydrolase activity. The substrate specificity of EndoS was also explored and showed that EndoS hydrolyses a broad range of glycans from the IgG scaffold. EndoS was also shown to have activity against alternative glycoprotein substrates, however, this non-specific activity was negligible in the context of whole serum. The effect of EndoS-mediated deglycosylation on the structure of the IgG Fc domain was explored using both X-ray crystallography and small-angle X-ray scattering. Small angle X-ray scattering was also used to characterise both EndoS and IdeS in complex with IgG Fc. Solution-state models of each complex were produced providing preliminary data towards how these enzymes interact with IgG. Overall, the results presented here contribute to our understanding of these enzymes which is of importance as they go forward into clinical applications.

Cerebral ischemia is a highly debilitating condition where shortage of oxygen and glucose leads to profuse cell death. Insufficient blood supply to the brain leads to cerebral ischemia and increase in extracellular lactate concentrations. Rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischemia. Lactate is a neuroprotective metabolite whose concentrations increase to 15-30 mM during ischemia and TREK1 is a neuroprotective potassium channel which is upregulated during ischemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischemia, however the connecting link between the two is missing. We hypothesized that lactate might interact with TREK1 channels and mediate neuroprotection. The aim of this study was to investigate the effect of lactate on activity and expression of TREK1 channels and evaluate the role of lactate-TREK1 interaction in conferring neuroprotection in the ischemia-prone hippocampus Ischemic concentrations (15-30 mM) of lactate at pH 7.4 increased whole cell TREK1 current in CA1 stratum radiator astrocytes and caused membrane hyperpolarization. We confirmed the intracellular action of lactate on TREK1 in hippocampal slices using mono carboxylate transporter blockers. The intracellular effect of lactate on TREK1 channels is specific since other mono carboxylates such as pyruvate at pH 7.4 failed to increase TREK1 current. We used immunostaining, western blot and electrophysiology to show that 15-30 mM of lactate increased functional TREK1 protein expression by 1.5-3 fold in hippocampal astrocytes. Next, we performed quantitative PCR to investigate if the increase in TREK1 protein expression was due to increased transcription and found that lactate stimulated TREK1 mRNA transcription to increase TREK1 protein. Lactate mediated increase in TREK1 expression was dependent on protein kinase A as inhibitors of protein kinase A abolished the increase in TREK1 mRNA and protein. The role of lactate-TREK1 interaction in neuroprotection was subsequently investigated using an in vitro oxygen glucose deprivation model of ischemia. Addition of 30 mM lactate to oxygen glucose deprived slices reduced neuronal death in the hippocampal CA1 pyramidal layer. However, 30 mM lactate failed to reduce cell death in rat hippocampal slices treated with TREK1 channel blockers signifying the requirement of active TREK1 channels for lactate mediated neuroprotection. However, lactate in the presence of protein kinase inhibitor failed to reduce cell death. This might be related to the role of protein kinase A in upregulation of TREK1 channels. We also estimated CA1 pyramidal neuronal TREK1 channel expression and found both lactate and oxygen glucose deprivation to decrease TREK1 channel expression that was surprisingly opposite to the effects on astrocytes. As TREK1 channel activation and upregulation decreases neuronal excitability, a decrease in neuronal TREK1 channel expression in response to lactate is expected to cause higher neuronal death and fails to explain lactate mediated neuroprotection. Since, lactate upregulated TREK1 channel expression and functional activity in CA1 stratum radiate astrocytes, we reasoned that the lactate mediated neuroprotection might be via astrocytic TREK1 channels requiring viable functional astrocytes. This was tested by disrupting astrocyte function using gliotoxin, and estimating cell death in oxygen glucose deprived hippocampal slices. Lactate failed to reduce cell death in presence of gliotoxin signifying the importance of viable astrocytes for lactate mediated neuroprotection. The above effects were specific to lactate as pyruvate failed to increase TREK1 expression and reduce cell death. TREK1 channels contribute to neuroprotection by enhancing potassium buffering and glutamate clearance capacity of astrocytes. We propose that lactate promotes neuronal survival in hippocampus by increasing TREK1 channel expression and activity in astrocytes during ischemia. This pathway serves as an alternate mechanism of neuroprotection.

Structural biology has become an important part of researching various diseases and drug development. In this thesis, I provide details on how I worked with approaches to structural characterization of a heteromeric GABA(A)R. These pentameric ligand gated ion channels take part in regulating inhibition of action potentials in nerve cells by allowing the passage of Cl- ions when bound by gamma-aminobutyric acid (GABA). They are formed by the assembly of five subunits which can be of various different types, denoted by greek letters and a number. Much is still unknown about how GABA and several other ligands bind to these ion channels and how that impacts function. Obtaining a structure of these proteins can aid in closing those knowledge gaps. It is reasonable to screen the proteins you have before you study their structures by Cryo-EM in order to get the best result, a methodology for which is described here. I have followed this methodology to screen two heteromeric GABA$_A$R that we wish to determine the structure of, alpha 5 beta 3 and rho 1 gamma 2. Neither of the combinations of genes we used to express these proteins proved to produce the desired fully assembled heteromeric protein. In the case of alpha 5 beta 3, we only witnessed building blocks, with no fully assembled channels. In rho 1 gamma 2, we instead only witnessed fully formed homomers of the rho 1 subunit. These findings then exclude the gene constructs used from further structural study, and the methodology described will inform the next steps to be taken. / Strukturbiologi har blivit en viktig del av forskningen kring många sjukdomar samt utveckling av läkemedel. I denna uppsats delger jag hur jag arbetat med metoder för strukturell karakterisering av en heteromerisk GABA(A)R. Dessa pentameriska ligandstyrda jonkanaler deltar i regleringen av hämning av aktionspotentialer i nervceller genom att tillåta passagen av Cl- joner när gamma-aminosmörsyra (GABA) binder. Dessa består av fem subenheter som kan vara en av flera olika typer, vilka anges med en grekisk bokstav och en siffra. Mycket om hur GABA och andra ligander binder till dessa jonkanaler och hur det påverkar dess funktion är fortfarande okänt. Att hitta en struktur av dessa proteiner kan hjälpa oss att stänga kunskapsgapen. Det är klokt att undersöka om genen man ska använda för att uttrycka det sökta proteinet ger det man söker innan man sen börjar studera strukturen. Jag har beskrivit en metodologi för detta och följt den för två heteromeriska proteiner, alpha 5 beta 3 och rho 1 gamma 2. Ingen av kombinationerna av gener vi använt för att uttrycka dessa proteiner har producerat de sökta, fullt ihoppbyggda proteinerna. I fallet för alpha 5 \beta 3 så ser vi endast byggstenar och inga kompletta proteiner, och för rho 1 gamma 2 så ser vi endast homomeriska proteiner av rho 1. Dessa slutsatser exkluderar de genkonstruktioner vi använt från vidare strukturella studier, och stegen som bör tas härnäst beskrivs av den använda metodologin.

X-ray free-electron lasers (XFELs) can achieve near-atomic resolution in imaging organic molecules. As a fourth-generation light source, modern XFELs can generate brilliant ultrashort X-ray pulses at MHz repetition rates. This allows XFELs to image single molecules with great detail, obtaining information about their dynamics and states through the interaction of the electrons within the molecule with the X-rays. A key challenge when imaging biomolecules (e.g. proteins, viruses, or bacteria) is to image the sample within its native environment, in solution. 3D-printed gas-accelerated liquid-sheet jet nozzles for liquid sample-delivery have yielded promising results in this respect, demonstrating that liquid sheets can be a reliable alternative to conventional sample-delivery methods, e.g. electrospray. Although the nozzles that this project uses have been successfully used for measurements at XFELs, the effect of nozzle design and liquid material-properties have not previously been explored. Therefore, the present report aims to explore different flow regimes of gas-accelerated liquid-sheet jets, and to study how the generated sheet jet depends upon different parameters, such as gas and liquid flow-rates, sample solution, and nozzle geometry. The findings suggest that low surface tension is crucial for producing large jets, and that higher viscosity may help to generate more stable sheet jets. However, further studies are required to draw definite conclusions.

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.