Integral Membrane Proteins (IMPs) are an important and scientifically interesting class of protein which span the lipid bilayer surrounding cells, cell compartments and many viruses. Molecular Dynamics (MD) simulation has revealed intimate and often highly specific relationships between membrane lipids and IMPs critical to many metabolic and signalling pathways. Meanwhile, the use of Coarse-Grained (CG) MD techniques has extended capabilities of biomolecular simulation to larger proteins over longer time periods. Several tools and resources for biomolecular simulations of IMPs are presented here, as well as two MD studies of specific IMPs. The previously developed MemProtMD pipeline automates the setup of MD simulations of IMPs; major extensions to this are presented here with the MemProtMD database and web server, automating the analysis of IMP simulations. The results of this can be viewed using the MemProtMD web server, an interactive, searchable online resource containing data from simulations of over 3000 experimentally determined IMP structures in explicit lipid bilayers. Using data from analysis of the entire MemProtMD database, MemProtMetrics has been developed to automate identification and orientation of IMP structures from Protein DataBank (PDB) depositions. This is shown to effectively predict membrane protein orientations seen in MD simulations. A tool for identification and classification of membrane lipids is also described, and used to identify over 500 IMPs structures with resolved lipids. CGMD simulations have also been used to assess dependence on side-chain ionisation state of interactions between lipids and two IMPs observed in mass spectrometry experiments. The simulations reveal similar trends to those seen in experiments. Finally, using multi-scale simulations, and through the development of a novel method for altering membrane composition in MD simulations, lipid-specific scramblase activity was shown for a novel structure of the TMEM16K scramblase IMP.

Ellen Lai Ping Chan. / Thesis (Ph.D)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 238-272. / Chapter CHAPTER I --- LITERATURE REVIEW / Chapter 1. --- History --- p.1 / Chapter 2. --- Species Distribution of BAT --- p.3 / Chapter 3. --- Distribution of BAT --- p.4 / Chapter 4. --- Structure of BAT --- p.4 / Chapter 4.1. --- Macroscopic Appearance --- p.4 / Chapter 4.1.1. --- Innervation --- p.4 / Chapter 4.1.2. --- Blood supply --- p.5 / Chapter 4.2. --- Microscopic Structure of BAT --- p.6 / Chapter 4.3. --- Difference Between Brown Fat and White Fat --- p.9 / Chapter 5. --- Composition of BAT --- p.11 / Chapter 6. --- The Mechanisms of Brown Adipose Tissue Thermogenesis --- p.12 / Chapter 6.1. --- Factors Influencing Proton Transport by UCP --- p.16 / Chapter 6.2. --- Postulated Sequence of Events during Thermogenesis --- p.18 / Chapter 7. --- Measurements of thermogenic Capacity of BAT --- p.21 / Chapter 8. --- Age-related Differences in BAT --- p.28 / Chapter 9. --- Non-shivering Thermogenesis and BAT --- p.32 / Chapter 9.1. --- Changes In BAT During Cold Acclimation --- p.35 / Chapter 9.1.1. --- GDP Binding --- p.35 / Chapter 9.1.2. --- Concentration of UCP --- p.37 / Chapter 9.1.3. --- Metabolic changes in BAT during Cold Acclimation --- p.39 / Chapter 10. --- Diet-induced Thermogenesis and BAT --- p.41 / Chapter 10.1. --- Mechanism of DIT --- p.42 / Chapter 10.2. --- Controversies in DIT --- p.44 / Chapter 10.3. --- Nutritional Factors Inducing DIT --- p.46 / Chapter 10.4. --- DIT in Man --- p.47 / Chapter 10.5. --- Neuroendocrine Control of BAT in DIT --- p.48 / Chapter 10.6. --- Effects of Fasting in BAT --- p.51 / Chapter 11. --- Obesity and BAT --- p.53 / Chapter 11.1. --- NST and DIT in Obese Animals --- p.58 / Chapter 11.2. --- Regulation of BAT in Obese Animals --- p.59 / Chapter 11.2.1. --- Sympathetic Nervous System in Obese Animals --- p.59 / Chapter 11.2.2. --- Corticosteriods in Obese Animals --- p.61 / Chapter 11.2.3. --- Adrenergic Receptors in Obese Animals --- p.63 / Chapter 11.2.4. --- Insulin in Obese Animals --- p.64 / Chapter 12. --- Pregnancy and Lactation and BAT --- p.67 / Chapter 12.1. --- Energy Balance During Pregnancy and Lactation --- p.67 / Chapter 12.2. --- Some Metabolic Changes During X Pregnancy and Lactation --- p.68 / Chapter 12.3. --- Role of BAT in Pregnancy and Lactation --- p.70 / Chapter 12.4. --- Mechanism of Regulation of Thermogenesis during Pregnancy and Lactation --- p.71 / Chapter 13. --- Factors Controlling the Thermogenesis --- p.75 / Chapter 13.1. --- Sympathetic Nervous Control --- p.75 / Chapter 13.1.1. --- Studies of Administration of Noradrenaline --- p.75 / Chapter 13.1.2. --- Control of the Fuel Supply to BAT by Sympathetic Nervous System --- p.77 / Chapter 13.1.3. --- Sympathetic denervation --- p.78 / Chapter 13.2. --- Hormonal Control --- p.79 / Chapter 13.2.1. --- Thyroid Hormone --- p.79 / Chapter 13.2.2. --- Insulin --- p.81 / Chapter 13.2.3. --- Pituitary Hormones --- p.83 / Chapter 13.2.4. --- Glucocorticoids --- p.83 / Chapter 13.2.5. --- Corticotropin-Releasing Factor --- p.85 / Chapter 14. --- Aims of the Study --- p.87 / Chapter CHAPTER II --- ISOLATION AND PURIFICATION OF UCP AND DEVELOPMENT OF AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR UCP / Chapter 1. --- INTRODUCTION --- p.88 / Chapter 2. --- MATERIALS AND METHODS --- p.89 / Chapter 2.1. --- Animals --- p.89 / Chapter 2.2. --- Collection of BAT --- p.89 / Chapter 2.3. --- Isolation of Mitochondria --- p.90 / Chapter 2.4. --- Electron Microscopy (EM) of Isolated BAT Mitochondria --- p.92 / Chapter 2.5. --- Measurement of Protein and Cytochrome C Oxidase Activity --- p.94 / Chapter 2.5.1. --- Measurement of Protein Concentration --- p.94 / Chapter 2.5.2. --- Measurement of Cytochrome C Oxidase Activity --- p.99 / Chapter 2.6. --- GDP Binding Assay of BAT Mitochondria --- p.101 / Chapter 2.6.1. --- GDP Binding Assay of Mitochondria by Centrifugation Method --- p.103 / Chapter 2.6.2. --- GDP: Binding Activity by Equilibrium Dialysis --- p.106 / Chapter 2.6.3. --- GDP Binding by Microfiltration Method --- p.108 / Chapter 2.7. --- Experiments Designed for Validation of GDP Binding Assay --- p.109 / Chapter 2.7.1. --- GDP Binding Activity in BAT Mitochondria after Noradrenaline Treatment --- p.109 / Chapter 2.7.2. --- GDP Binding Activity in BAT Mitochondria after Cold Acclimation and Noradrenaline Treatment --- p.110 / Chapter 2.7.3. --- Effect of Food Restriction on Cold Acclimated Rats --- p.110 / Chapter 2.7.4. --- GDP Binding Activity of BAT Mitochondria of Rats of Different Ages --- p.111 / Chapter 2.8. --- Isolation and Purification of UCP --- p.111 / Chapter 2.9. --- Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.115 / Chapter 2.10. --- Methods for Raising Anti-Rat-UCP Antibody and the Characterization of Antiserum --- p.120 / Chapter 2.10.1. --- Raising Rabbit Anti-Rat-UCP Antibody --- p.120 / Chapter 2.10.2. --- Western Blot Analysis For Cross Reactivity Study --- p.120 / Chapter 2.10.3. --- Immuno-Autoradiographic Method for Detection of Specificity of Rabbit Anti-Rat UCP Antiserum --- p.121 / Chapter 2.11. --- Enzyme Linked Immunosorbent Assay For UCP --- p.124 / Chapter 2.12. --- Experiment Designed to Validate the ELISA --- p.129 / Chapter 2.13. --- Statistical Analysis --- p.129 / Chapter 3. --- RESULTS --- p.130 / Chapter 3.1. --- Electron Microscopy of Isolated BAT Mitochondria --- p.130 / Chapter 3.2. --- GDP Binding Assay of BAT Mitochondria --- p.130 / Chapter 3.3. --- Experiments Designed for Validation of GDP Binding Assay --- p.133 / Chapter 3.3.1. --- GDP Binding Activity of BAT Mitochondria after Noradrenaline Injection --- p.133 / Chapter 3.3.2. --- GDP Binding Activity of BAT Mitochondria after Cold Acclimation and Noradrenaline Treatment --- p.136 / Chapter 3.3.3. --- Effects of Food Restriction on Cold Acclimated Rats --- p.136 / Chapter 3.3.4. --- GDP Binding Activity of BAT Mitochondria from Rats of Different Ages --- p.140 / Chapter 3.4. --- Isolation and Purification of UCP --- p.140 / Chapter 3.4.1. --- Results of SDS-PAGE --- p.143 / Chapter 3.4.2. --- Results of GDP Binding Activity --- p.149 / Chapter 3.5. --- Rabbit Anti-rat-UCP Antibody and the Characterization of Antiserum --- p.151 / Chapter 3.5.1. --- Immuno-autoradiography for Specificity of Rabbit Anti-rat-UCP Antiserum --- p.153 / Chapter 3.5.1.1. --- Cross-reactivity of the Rabbit Anti-rat-UCP Antiserum to Mitochondrial Proteins of BAT and from other Tissues --- p.153 / Chapter 3.5.1.2. --- Cross-reactivity of the Rabbit Anti-rat-UCP Antiserum to BAT Mitochondrial Protein from Different Rodent Species --- p.156 / Chapter 3.5.1.3. --- Dose Response of Rabbit Anti-rat-UCP Antiserum to UCP --- p.159 / Chapter 3.6. --- ELISA of UCP --- p.161 / Chapter 3.6.1. --- Determination of Maximum Amount of UCP Binding on Microtitre Plate --- p.161 / Chapter 3.6.2. --- Antibody Dilution Curve --- p.161 / Chapter 3.6.3. --- Incubation Time for Enzyme-Substrate Reaction --- p.163 / Chapter 3.6.4. --- Competitive ELISA --- p.163 / Chapter 3.6.5. --- Precision of ELISA --- p.167 / Chapter 3.7. --- Experiment Designed for Validation of ELISA by Measuring UCP in Cold Acclimated Rats --- p.170 / Chapter 4. --- DISCUSSION --- p.172 / Chapter 4.1. --- GDP Binding Assay of BAT Mitochondria --- p.172 / Chapter 4.2. --- Isolation and Purification of UCP --- p.176 / Chapter 4.3. --- Development and Evaluation of ELISA --- p.178 / Chapter CHAPTER III --- CHANGES IN BAT DURING PREGNANCY AND LACTATION AND ROLE OF PROLACTIN / Chapter 1. --- INTRODUCTION --- p.184 / Chapter 2. --- MATERIALS AND METHODS --- p.187 / Chapter 2.1. --- Animal --- p.187 / Chapter 2.2. --- Experimental Designs --- p.187 / Chapter 2.2.1. --- "Effects of Pregnancy, Lactation and Post Weaning on BAT" --- p.187 / Chapter 2.2.2. --- Effect of Metoclopramide on BAT --- p.188 / Chapter 2.2.3. --- Effect of Metoclopramide and Bromocriptine on BAT --- p.188 / Chapter 2.2.4. --- Effect of PRL Injection on BAT --- p.189 / Chapter 2.2.5. --- Continuous infusion of PRL --- p.189 / Chapter 2.6.6. --- Measurements of BAT Parameters --- p.191 / Chapter 2.2.7. --- RIA of serrum PRL --- p.191 / Chapter 2.2.8. --- PRL Receptors in BAT --- p.197 / Chapter 2.4. --- Statistical Analysis --- p.201 / Chapter 3. --- RESULTS --- p.202 / Chapter 3.1. --- Effects of Pregnancy and Lactation --- p.202 / Chapter 3.1.1. --- Food Consumption and Body Weight --- p.202 / Chapter 3.1.2. --- BAT --- p.205 / Chapter 3.1.3. --- Serum PRL level --- p.209 / Chapter 3.2. --- Effects of PRL njection --- p.213 / Chapter 3.3. --- Effects of Continuous Infusion of PRL on BAT --- p.213 / Chapter 3.4. --- Effects of Metoclopramide on BAT --- p.216 / Chapter 3.5. --- Effects of Bromocriptine and Metoclopramide on BAT --- p.216 / Chapter 3.6. --- PRL Receptor in BAT --- p.219 / Chapter 4. --- DISCUSSION --- p.223 / GENERAL CONCLUSION --- p.236

Many Gram-positive and Gram-negative bacteria produce an additional thick layer of carbohydrate polymers on the cell wall surface. These capsules (capsular polysaccharides; CPS) play critical roles in interactions between bacteria and their environments (Whitfield, 2006). This is especially important in infection processes since for both Gram-negative and Gram-positive pathogens CPS is the point of first contact with the host immune system (Whitfield, 2006). However, the details of CPS biosynthesis and assembly mechanisms are still unclear. Therefore, we embarked on structural and kinetic studies of the proteins Wzc, Wza and Wzb/ Cps4B from the Wzy-dependent pathway, as well as the protein WbdD from the ATP-binding cassette (ABC) transporter dependent system. Full-length Wzc failed to crystallise due to the presence of large disordered regions and the overall difficulty of membrane protein crystallisation. A truncated version of Wzc (1-480) without the C-terminal tyrosine kinase domain was crystallised and diffracted to 15 Å in house. A previous study suggested Wza and Wzc form a functional complex (Whitfield, 2006), so Wza was also studied. Since the full-length Wza structure is available (C. Dong et al., 2006), Pulsed electron–electron double resonance spectroscopy (PELDOR) was used to study the conformational change. The PELDOR spectroscopy distance fingerprint of Wza was determined. These data also confirmed that PELDOR is a powerful tool to study large, highly symmetrical membrane proteins and can be used to study other complex membrane protein systems, such as ion channels or transporters. The crystal structure of Wzb the cognate phosphatase of Wzc was determined to 2.2 Å. Also Cps4B, which is a functional homologue of Wzb but has a completely unrelated sequence, was crystallised in two crystal forms. Form I and II Cps4B crystals diffracted to 2.8 Å and 1.9 Å resolution in house, respectively. The full-length WbdD failed to crystallise due to the presence of large disordered regions. Therefore, a shorter construct, WbdD₅₅₆ (1-556) was cloned and crystallised. The structure was determined to 2.2 Å. WbdD is a bifunctional enzyme consisting of a methyltransferase (MTase) and a kinase domain. In order to better understand the function of this protein, a variety of techniques were used, such as the ADP-Glo kinase assay, Nuclear magnetic resonance (NMR) spectroscopy, small angle X-ray scattering (SAXS) and X-ray crystallography. The various findings in the current projects provide meaningful insights towards a better understanding of the CPS biosynthesis and assembly mechanisms, which may contribute to a more intensive study identifying inhibitors and beginning to unravel the mechanism of chain length regulation.

Genomic analysis is a new approach for the characterization and investigation of novel genes, gene clusters, the function of uncharacterized proteins, and genetic diversity in microorganisms. These approaches are important for the study of chlamydiae, a system in which several genomes have been sequenced but in which techniques for genetic manipulation are not available. The objective of this thesis is to combine computer-based analysis of chiamydial inclusion membrane proteins (Incs) with cellular and molecular biological analysis of the bacteria. Three different experimental lines of investigation were examined, focusing on Incs of C. trachomatis and C. pneumoniae. Chlamydiae are obligate intracellular bacteria that develop within a nonacidified membrane bound vacuole termed an inclusion. Putative Inc proteins of C. trachomatis and C. pneumoniae were identified from genomic analysis and a unique structural motif. Selected putative Inc proteins are shown to localize to the inclusion membrane. Chiamydia trachomatis variants with unusual multiple-lobed, nonfusogenic, inclusion were identified from a large scale serotyping study. Fluorescence microscopy showed that IncA, a chiamydial protein localized to the inclusion membrane, was undetectable on non-fusogenic inclusions of these variants. Sequence analysis of incA from non-fusogenic variant isolates revealed a defective incA in most of the variants. Some variants lack not only IncA on the inclusion membrane but also CT223p, an additional Inc protein. However, no correlation between the absence of CT223p and distinctive inclusion phenotype was identified. Nucleotide sequence analysis revealed sequence variations of C. trachomatis incA and CT223 in some variant and wild type isolates. Comparative analyses of the three recently published C. pneumoniae genomes have led to the identification of a novel gene cluster named the CPn1O54 gene family. Each member of this family encodes a polypeptide with a hydrophobic domain characteristic of proteins localized to the inclusion membrane. These studies provided evidence that gene variation might occur within this single collection of paralogous genes. Collectively, the variability within this gene family may modulate either phase or antigenic variation, and subsequent physiologic diversity, within a C. pneumoniae population. These studies demonstrate the genetic diversity of Inc proteins and candidate Inc proteins, within and among the different chiamydial species. This work sets the stage for further investigations of the structure and function of this set of proteins that are likely critical to chlamydial intracellular growth. / Graduation date: 2003

There is no abstract available for this thesis. / Department of Chemistry

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 200-212. / Introduction -- Shotgun proteomic analysis of rat liver membrane proteins -- A combination of immobilised pH gradients improve membrane proteomics -- Affects of tumor-induced inflammation on membrane proteins abundance in the mouse liver -- Affects of tumor-induced inflammation on biochemical pathways in the mouse liver -- General discussion -- References. / The aim of this thesis was to develop a proteomics methodology that improves the identification of membrane proteomes from mammalian liver. Shotgun proteomics is a method that allows the analysis of proteins from cells, tissues and organs and provides comprehensive characterisation of proteomes of interest. The method developed in this thesis uses separation of peptides from trypsin digested membrane proteins by immobilised pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two dimensional shotgun proteomics. In this thesis, peptide IPG-IEF was shown to be a highly reproducible, high resolution analytical separation that provided the identification of over 4,000 individual protein identifications from rat liver membrane samples. Furthermore, this shotgun proteomics strategy provided the identification of approximately 1,100 integral membrane proteins from the rat liver. The advantages of using peptide IPG-IEF as a shotgun proteomics separation dimension in conjunction with label-free quantification was applied to a biological question: namely, does the presence of a spatially unrelated benign tumor affect the abundance of mouse liver proteins. IPG-IEF shotgun proteomics provided comprehensive coverage of the mouse liver membrane proteome with 1,569 quantified proteins. In addition, the presence of an Englebreth-Holm-Swarm sarcoma induced changes in abundance of proteins in the mouse liver, including many integral membrane proteins. Changes in the abundance of liver proteins was observed in key liver metabolic processes such as fatty acid metabolism, fatty acid transport, xenobiotic metabolism and clearance. These results provide compelling evidence that the developed shotgun proteomics methodology allows for the comprehensive analysis of mammalian liver membrane proteins and detailed some of the underlying changes in liver metabolism induced by the presence of a tumor. This model may reflect changes that could occur in the livers of cancer patients and has implications for drug treatments. / Mode of access: World Wide Web. / 609 p. ill. (some col.)

O gene PKHD1, mutado na doença renal policística autossômica recessiva, apresenta um padrão de splicing complexo associado a múltiplos transcritos alternativos. Neste trabalho estudamos o perfil de expressão de seu produto, poliductina. Análises por western blot revelaram produtos putativos de membrana de > 440 kDa e ~230 kDa, e de ~140 kDa em frações solúveis de rim, fígado e pâncreas. Estudos imunoistoquímicos mostraram marcação em ductos coletores renais e porção ascendente espessa da alça de Henle, em epitélios ductais biliar e pancreático e, no período embrionário, em broto ureteral, ductos biliar e pancreático e glândula salivar. Análises por imunofluorescência e microscopia imunoeletrônica sugerem que poliductina se localize em cílio apical primário, membrana apical e citoplasma de células do ducto coletor. Nossos resultados indicam que PKHD1 codifica isoformas de membrana e solúveis / PKHD1, the gene mutated in autosomal recessive polycystic kidney disease, presents a complex splicing pattern, associated with multiple alternative transcripts. In this work we have studied the expression profile of its product, polyductin. Western blot analysis revealed putative membrane products of > 440 kDa and 230 kDa, and of ~140 kDa in soluble fractions in kidney, liver and pancreas. Immunohistochemistry studies showed staining in renal collecting duct and thick ascending limb of Henle, in biliary and pancreatic ductal epithelia and, in the embryonic period, in ureteric bud, biliary and pancreatic ducts and salivary gland. Immunofluorescence and immunoelectron microscopy studies suggest that polyductin localizes to primary apical cilium, apical membrane and cytoplasm of collecting duct cells. Our data indicate that PKHD1 codifies membrane and soluble isoforms

O gene PKHD1, mutado na doença renal policística autossômica recessiva, apresenta um padrão de splicing complexo associado a múltiplos transcritos alternativos. Neste trabalho estudamos o perfil de expressão de seu produto, poliductina. Análises por western blot revelaram produtos putativos de membrana de > 440 kDa e ~230 kDa, e de ~140 kDa em frações solúveis de rim, fígado e pâncreas. Estudos imunoistoquímicos mostraram marcação em ductos coletores renais e porção ascendente espessa da alça de Henle, em epitélios ductais biliar e pancreático e, no período embrionário, em broto ureteral, ductos biliar e pancreático e glândula salivar. Análises por imunofluorescência e microscopia imunoeletrônica sugerem que poliductina se localize em cílio apical primário, membrana apical e citoplasma de células do ducto coletor. Nossos resultados indicam que PKHD1 codifica isoformas de membrana e solúveis / PKHD1, the gene mutated in autosomal recessive polycystic kidney disease, presents a complex splicing pattern, associated with multiple alternative transcripts. In this work we have studied the expression profile of its product, polyductin. Western blot analysis revealed putative membrane products of > 440 kDa and 230 kDa, and of ~140 kDa in soluble fractions in kidney, liver and pancreas. Immunohistochemistry studies showed staining in renal collecting duct and thick ascending limb of Henle, in biliary and pancreatic ductal epithelia and, in the embryonic period, in ureteric bud, biliary and pancreatic ducts and salivary gland. Immunofluorescence and immunoelectron microscopy studies suggest that polyductin localizes to primary apical cilium, apical membrane and cytoplasm of collecting duct cells. Our data indicate that PKHD1 codifies membrane and soluble isoforms