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On the quantitative analysis of electronic energy transfer/migration in proteins studied by fluorescence spectroscopyIsaksson, Mikael January 2007 (has links)
Two recently developed theories of electronic energy transfer/migration were for the first time applied to real protein systems for extracting molecular distances. The partial donor-donor energy migration (PDDEM) is an extension to the previously developed donor-donor energy migration (DDEM, F Bergström et al PNAS 96, 1999, 12477) which allows using chemically identical but photophysically different fluorophores in energy migration experiments. A method based on fluorescence quenching was investigated and applied to create an asymmetric energy migration between fluorophores which were covalently and specifically attached to plasminogen activator inhibitor type 2 (PAI-2). It was also shown experimentally that distance information can be obtained if the fluorescence relaxation for photophysically identical donors, exhibits multi-exponential relaxation. An extended Förster theory (EFT) that was previously derived (L. B.-Å. Johansson et al J. Chem. Phys., 1996, 105) ha been developed for analysis of donor-acceptor energy transfer systems as well as DDEM systems. Recently the EFT was also applied to determine intra molecular distances in the protein plasminogen activator inhibitor type 1 (PAI-1) which was labelled with a sulfhydryl specific derivative of BODIPY. The EFT explicitly accounts for the time-dependent reorientations which in a complex manner influence the rate of electronic energy transfer/migration. This difficulty is related to the “k2-problem”, which has been solved. It is also shown experimentally that the time-correlated single-photon counting (TCSPC) data is sensitive to the mutual configuration between the interacting fluorophores. To increase the accuracy in the extracted parameters it is furthermore suggested to collect the fluorescence data under various physico-chemical conditions. It was also shown that the Förster theory is only valid in the initial part of the fluorescence decay.
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Protein structure dynamics and interplay : by single-particle electron microscopyElmlund, Hans January 2008 (has links)
Single-particle cryo-electron microscopy (cryo-EM) is a method capable of obtaining information about the structural organization and dynamics of large macromolecular assemblies. In the late nineties, the method was suggested to have the potential of generating “atomic resolution” reconstructions of particles above a certain mass. However, visualization of secondary structure elements in cryo-EM reconstructions has so far been achieved mainly for highly symmetrical macromolecular assemblies or by using previously existing X-ray structures to solve the initial alignment problem. A factor that severely limits the resolution for low-symmetry (point group symmetry Cn) particles is the problem of ab initio three-dimensional alignment of cryo-EM projection images of proteins in vitreous ice. A more general problem in the field of molecular biology is the study of heterogeneous structural properties of particles in preparations of purified macromolecular complexes. If not resolved, structural heterogeneity limits the achievable resolution of a cryo-EM reconstruction and makes correct biological interpretation difficult. If resolved, the heterogeneity instead offers a tremendous biological insight into the dynamic behaviour of a structure, and statistical information about partitioning over subpopulations with distinct structural features within the ensemble of particles may be gained. This thesis adds to the existing body of methods in the field of single-particle cryo-EM by addressing the problem of ab initio rotational alignment and the problem of resolving structural heterogeneity without using a priori information about the structural variability within large populations of cryo-EM projections of unstained proteins. The thesis aims at making the single-particle cryo-EM method a generally applicable tool for generating subnanometer resolution reconstructions and perform heterogeneity analysis of biological macromolecules. / QC 20100719
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Imaging lipid phase separation in droplet interface bilayersDanial, John Shokri Hanna January 2015 (has links)
The spatiotemporal organization of membrane proteins is implicated in cellular trafficking, signalling and reception. It was proposed that biological membranes partition into lipid rafts that can promote and control the organization of membrane proteins to localize the mentioned processes. Lipid rafts are thought to be transient (microseconds) and small (nanometers), rendering their detection a challenging task. To circumvent this problem, multi-component artificial membrane systems are deployed to study the segregation of lipids at longer time and length scales. In this thesis, multi-component Droplet Interface Bilayers (DIBs) were imaged using fluorescence and interferometric scattering microscopy. DIBs were used to examine and manipulate microscopic lipid domains and to observe, for the first time, transient nanoscopic lipid domains. The techniques and results described here will have important implications on future research in this field.
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Engineered α-hemolysin pores with chemically and genetically-fused functional proteinsMantri, Shiksha January 2013 (has links)
Protein engineering could be used to bring two proteins together, which don't normally interact, in an oriented configuration. Using computer modelling and experimental work involving mutagenesis, a new dimer complex, (α7)2, was engineered with two α-hemolysin (αHL) heptamers (α7) units linked via disulfide bridges in a cap-to-cap orientation. The structure of (α7)2 was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. Importantly, it was shown that the one of two transmembrane barrels of (α7)2 can insert into an attoliter liposome, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM. In potential, (α7)2 could be used for small molecule transfer between micron-sized vesicles (minimal cells) and would have applications in forming proto-tissues from minimal cells. Another target has been to couple a highly processive exonuclease, λ-exonuclease (λ-exo), which functions as a trimer, with the α7 pore for DNA sequencing and single molecule studies of λ-exo. Several genetic fusion constructs of λ-exo and αHL were screened and optimized for activity. By linking the N-terminus of λ-exo monomer to the C-terminus of the αHL monomer (α1), a new kind of processive exonuclease (AE) was synthesized that can form pores in bilayers. AE and wild-type α1 could be integrated into hetero-heptamers with different number of AE subunits. To achieve a hetero-heptamer with only one λ-exo trimer molecule mounted on the αHL cap, a concatemer of 2 λ-exo (exo3) was made by genetically linking the monomers of λ-exo with 15 and 17 amino acid linkers. The immediate next step is to link exo3 to α1 and then to co-assemble the exo3-α1 fusion construct with α1 to make the λ-exo-αHL pore complex. Using similar strategies as described in this thesis, other proteins could be linked to αHL increasing the scope of the nanopore technology.
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Cloning, overexpression and biophysical characterization of grd/grl/wrt domains from<em> Caenorhabditis elegans</em> in<em> Escherichia coli</em>Lindberg, Marie January 2008 (has links)
<p>Hedgehog related genes have been shown to play a major role in development in all deuterostomes. In C.elegans, such genes have been found where the similarity is restricted to the C-terminal domain. This work has focused on the hedgehog related C.elegans proteins called ground (grd), ground-like (grl), and wart (wrt) which appear to form a unique structural family.These proteins are cysteine rich and have conserved cysteine patterns which, together with thethought that they are secreted, are expected to be in disulfide form. Since the extracellular environment is very oxidizing and due to the conserved cysteine pattern, disulfide bonds are thought to play a big part in the folding and stabilization of these proteins. The stability of the protein and the formation of a disulfide bond are related through a thermodynamic cycle, which insures that the stabilization of the protein by the disulfide is reflected by the identical stabilization of the disulfide by the protein. Practically, there are numerous parameters that can be used to try to achieve the correct disulfide bonds and folding, when doing in vitro trials, some of which were used in this project. C.elegans proteins grd-5, grd-13, grl-24, wrt-3 and wrt-5 were studied in this project. All of the proteins were expressed and purified with success, with theexception of grl-24. All constructs formed inclusion bodies. Some refolding attempts were performed on grd-13 and wrt-3. The presence of a disulfide bond in refolded grd-13 was demonstrated using chemical fragmentation. In general, these attempts did not give correctly folded proteins but provide a foundation to continue experiments aimed at producing a native-like protein for structural and functional studies.</p>
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Infrared spectroscopy : Method development and ligand binding studiesKumar, Saroj January 2010 (has links)
Infrared spectroscopy detects molecular vibrations and assesses the properties of molecules and their environment. It is a powerful technique to detect ligand induced changes in biomolecules as it has distinct signals and provides different levels of structural information. An addition of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. It facilitates to study ligand binding of substrates, activators, inhibitors and ions to macromolecules as well as effect of pH, ionic strength or denaturants on the structure of macromolecules, which play an important role in drug development. This method was tested with two proteins cyt c and calcium ATPase. We studied phosphoenol pyruvate (PEP) in different ionization states by infrared spectroscopy combined with theoretical analysis. Theoretical calculations helped to assign the bands. The infrared spectrum of labeled PEP and infrared measurement in D2O also helped in band assignment. We used the method dialysis accessory to attenuated total reflection infrared spectroscopy to investigate the binding of PEP and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. Isotopic labeled PEP helped to assign and evaluate the infrared absorption bands. The difference spectrum of bound and free PEP indicates specific interactions between ligand and protein. The quantitative evaluation revealed that the enzyme environment has little influence on the P-O bond strengths, which are weakened by less than 3% upon binding. The carboxylate absorption bands indicate shortening of the C-O bond by as little as 1.3 pm. The binding of PEP to PK in presence of monovalent cations K+ and Na+ showed that the binding interactions are very similar. / doctoral
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Cloning, overexpression and biophysical characterization of grd/grl/wrt domains from Caenorhabditis elegans in Escherichia coliLindberg, Marie January 2008 (has links)
Hedgehog related genes have been shown to play a major role in development in all deuterostomes. In C.elegans, such genes have been found where the similarity is restricted to the C-terminal domain. This work has focused on the hedgehog related C.elegans proteins called ground (grd), ground-like (grl), and wart (wrt) which appear to form a unique structural family.These proteins are cysteine rich and have conserved cysteine patterns which, together with thethought that they are secreted, are expected to be in disulfide form. Since the extracellular environment is very oxidizing and due to the conserved cysteine pattern, disulfide bonds are thought to play a big part in the folding and stabilization of these proteins. The stability of the protein and the formation of a disulfide bond are related through a thermodynamic cycle, which insures that the stabilization of the protein by the disulfide is reflected by the identical stabilization of the disulfide by the protein. Practically, there are numerous parameters that can be used to try to achieve the correct disulfide bonds and folding, when doing in vitro trials, some of which were used in this project. C.elegans proteins grd-5, grd-13, grl-24, wrt-3 and wrt-5 were studied in this project. All of the proteins were expressed and purified with success, with theexception of grl-24. All constructs formed inclusion bodies. Some refolding attempts were performed on grd-13 and wrt-3. The presence of a disulfide bond in refolded grd-13 was demonstrated using chemical fragmentation. In general, these attempts did not give correctly folded proteins but provide a foundation to continue experiments aimed at producing a native-like protein for structural and functional studies.
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Protein complexes in the gas phase : structural insights from ion mobility-mass spectrometry and computational modellingHall, Zoe Lauren January 2013 (has links)
Structure determination of macromolecular protein assemblies remains a challenge for well-established experimental methods. In this thesis, an emerging structural technique, ion mobility-mass spectrometry (IM-MS) is explored. An assessment of collision cross section (CCS) measurement accuracy using travelling-wave IM (TWIMS) instrumentation was carried out (Chapter 3). Through the collation of a protein complex CCS database and the development of a calibration framework for TWIMS, significant improvements to CCS measurement accuracy have been achieved. Next, the advantages and limitations of using IM-MS to generate restraints for structure characterisation were explored. Computational tools designed to exploit IM-MS data for structural modelling were developed and tested on a training set of systems (Chapter 4). These include two heteromeric protein complexes, and an oligomeric intermediate involved in beta-2-microglobulin aggregation. Further structural information can be attained by using gas-phase dissociation techniques, such as collision-induced dissociation (CID). The effects of charge state on CCS and the gas-phase dissociation pathway of complexes were investigated (Chapter 5). This highlighted the possibility of using CID in conjunction with supercharging to manipulate dissociation pathways to achieve more useful structural information. Finally, the gas-phase structures of globular and intrinsically disordered protein complexes were probed by IM-MS and molecular dynamics (MD) simulations (Chapter 6). Experimental observations were recapitulated remarkably closely by simulations, including gas-phase structural collapse and the ejection of monomer subunits when the energy of the system was increased sufficiently. Overall, this research has contributed to the IM-MS field by providing the framework for improved CCS measurements of large protein complexes and the use of restraints from IM-MS for structural modelling. Significantly, IM-MS has been used in combination with charge manipulation, CID and MD simulations to reveal further insights into the gas-phase structures, stabilities and dissociation pathways of multimeric protein complexes.
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Structural insights into membrane proteins, membrane protein-lipid interactions and drug metabolites in the gas-phase from ion mobility mass spectrometryReading, Eamonn January 2014 (has links)
Investigating the structures of membrane proteins and their interactions with lipids remains challenging for well-established biophysical techniques. In this thesis the use of mass spectrometry (MS) and ion mobility (IM) spectrometry were explored for the interrogation of membrane proteins, their stoichiometry, stability and interactions with lipids. The techniques used were also applied to the identification of drug metabolites. In the first two chapters reviews of both mass spectrometry methods, and membrane protein biogenesis and membrane protein-lipid interactions are presented. The first challenge for studying membrane proteins by MS was to optimise solution conditions. A detergent screening strategy was developed for this purpose (Chapter 3). The various detergent environments studied revealed dramatic differences in mass spectral quality permitting investigation of membrane protein-lipid interactions. Changes were observed in the electrospray charging of membrane proteins and trends were established from an extensive collection of membrane proteins ejected from a wide variety of detergent environments. The physicochemical principles behind the MS of membrane proteins were deduced and are presented (Chapter 4). The results of these experiments led to a deeper understanding of the ionisation processes and the influence of detergent micelles on both charge state and release mechanisms. Experiments from a range of different micelles also allowed the influence of charge and its effects on the preservation of native-like membrane protein conformations to be monitored by IM-MS. By resolving lipid-protein interactions, and by monitoring the effects of lipid binding on the unfolding of three diverse membrane protein complexes, substantial differences in the selectivity of membrane proteins for different lipids were revealed (Chapter 5). Interestingly lipids that stabilised membrane proteins in the gas-phase were found to induce modifications in structure or function thus providing an approach to assess direct lipid contributions, and to rank order lipids based on their ability to modulate membrane proteins. Using the MS approaches developed here also enabled study of the diversity of oligomeric states of the mechanosensitive channel of large conductance (MscL) (Chapter 6). Results revealed that the oligomeric state of MscL is sensitive to deletions in its C-terminal domain and to its detergent-lipid environment. Additionally, a case study with GlakoSmithKline (GSK) was undertaken using IM-MS technology but in this case applied to the identification of drug metabolites (Chapter 7). The results showed that IM-MS and molecular modelling could inform on the identity of different drug metabolites and highlights the potential of this approach in understanding the structure of various drug metabolites.
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Single-molecule chemistry studies with engineered alpha-hemolysin poresHammerstein, Anne Friederike January 2011 (has links)
Engineered protein nanopores can be used to investigate a wide range of dynamic processes in real time and at the single-molecule level, for example covalent bond making and breaking or the interaction of ligands with their cognate binding sites. The detection of such processes is accomplished by monitoring the current carried by ions through the pore in an applied potential, which is modulated as molecules of interest interact with engineered binding sites within the pore. In contrast to ensemble measurements, where the behaviour of individual molecules is obscured by averaging, single-channel recordings can identify short-lived intermediates and rare reaction pathways, thereby adding to our understanding of fundamental processes in chemistry and biology. The goal of my thesis work was to engineer alpha-hemolysin (αHL) pores to gain insight into such processes. <b>Chapter 1</b> provides an overview of common techniques used to study single- molecule processes, in particular single channel recordings. General techniques to engineer ion channels and pores are presented, followed by examples of how the alpha-HL pore has been engineered to monitor dynamic processes at the single- molecule level. <b>Chapter 2</b> describes how alpha-HL pores can be chemically modifeed with a tridentate "half-chelator" ligand. Single channel recordings show that this modifeed pore can be used to determine rates of chelation and the stability of divalent metal ion complexes. The modifeed pore can also be used as a stochastic sensor for the detection of different divalent metal ions in solution. <b>Chapter 3</b> investigates the chelate-cooperativity between two half-chelator ligands installed in close proximity in the alpha-HL pore, as they form a full complex with a single Zn<sup>2+</sup> ion. The single channel recordings reveal a two step process, in which the Zn<sup>2+</sup> ion must fiferst bind to one of the two half-chelators, before the second one completes the complex. The rate constants for all the major steps of the process are determined and the extent of cooperativity between the half-chelators is quantifeed. <b>Chapter 4</b> demonstrates that genetically encoded subunit dimers of alpha-HL can be used to control the subunit arrangement in the heptameric pore. Although techniques exist to prepare heteroheptameric pores, pores containing more than one type of modifeed subunit are not commonly used because it is impossible to distinguish between the permutations of the pore. By using subunit dimers, heptamers in which two defefined subunits are adjacent to each other can be formed, which increases the range of structures that can be obtained from engineered protein nanopores. <b>Chapter 5</b> explores the possibility of following the nuclease activity of a metal complex in the alpha-HL pore at the single-molecule level. The Rh(III) complex [Rh(bpy)2phzi]<sup>2+</sup> binds strongly to CC mismatches in dsDNA, and on activation with UV light promotes the cleavage of one of the two strands. To follow this reaction by single channel recording, a piece of dsDNA with the bound Rh-complex was immobilised in the HL pore and the single current changes under UV irradiation were monitored. The preliminary data indicate that the rate of the photocleavage reaction can be measured.
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