Lau Lai Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 125-141). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iii / Acknowledgement --- p.v / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Research initiative and significance --- p.1 / Chapter 1.2 --- Terminal differentiation --- p.4 / Chapter 1.3 --- Controversial terminal differentiation in cardiomyocytes --- p.5 / Chapter 1.4 --- Molecular switch from hyperplasia to hypertrophy in neonatal myocardial development --- p.7 / Chapter 1.5 --- Interferons --- p.8 / Chapter 1.6 --- Functions induced by interferons --- p.9 / Chapter 1.7 --- Interferons in cardiomyocytes --- p.12 / Chapter 1.8 --- Interferon-inducible transmembrane gene family --- p.13 / Chapter 1.9 --- Our hypothesis and objective --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Sequence analysis --- p.18 / Chapter 2.2 --- Cell culture --- p.18 / Chapter 2.3 --- Induction of differentiation of H9C2 cells --- p.19 / Chapter 2.4 --- In vitro induction of IFITMs by interferon treatments --- p.19 / Chapter 2.5 --- RNA isolation --- p.20 / Chapter 2.5.1 --- Experimental animals and sampling --- p.20 / Chapter 2.5.2 --- Total RNA Isolation --- p.20 / Chapter 2.5.3 --- RNA Quantification and Quality Check --- p.21 / Chapter 2.5.4 --- Purification by Qiagen-RNeasy Column and DNase I Digestion --- p.21 / Chapter 2.6 --- First-strand cDNA synthesis --- p.22 / Chapter 2.7 --- Quantitative real-time polymerase chain reaction --- p.22 / Chapter 2.8 --- Cloning protocol --- p.25 / Chapter 2.8.1 --- "Construction of pEGFP-IFITMl, pEGFP-IFITM2 and pEGFP-IFITM3 fusion proteins" --- p.25 / Chapter 2.8.1.1 --- Amplification of DNA fragments --- p.25 / Chapter 2.8.1.2 --- Purification of PCR product --- p.26 / Chapter 2.8.1.3 --- Restriction endonuclease digestion --- p.26 / Chapter 2.8.1.4 --- Insert/vector ligation --- p.27 / Chapter 2.8.1.5 --- Preparation of chemically competent bacterial cells --- p.27 / Chapter 2.8.1.6 --- Transformation of ligation product into chemically competent bacterial cells DH5a --- p.28 / Chapter 2.8.1.7 --- Recombinant clone screening by PCR --- p.29 / Chapter 2.8.1.8 --- Small-scale preparation of recombinant plasmid DNA --- p.29 / Chapter 2.8.1.9 --- Dideoxy DNA sequencing --- p.30 / Chapter 2.8.1.10 --- Large-scale preparation of recombinant plasmid DNA --- p.30 / Chapter 2.8.2 --- "Construction of IFITMl-pcDNA4, IFITM2-pcDNA4 and IFITM3- pcDNA4 constructs" --- p.33 / Chapter 2.8.2.1 --- Amplification of DNA fragments --- p.33 / Chapter 2.8.2.2 --- Insert/vector ligation --- p.33 / Chapter 2.8.2.3 --- Transformation of ligation product into one shot® TOP1 OF´ة chemically competent E. coli cells --- p.34 / Chapter 2.9 --- Transient transfection --- p.36 / Chapter 2.10 --- Subcellular fractionation --- p.37 / Chapter 2.11 --- Isolation of total protein cell lysate --- p.38 / Chapter 2.12 --- Protein concentration determination --- p.38 / Chapter 2.13 --- Protein gel electrophoresis and western blotting --- p.39 / Chapter 2.13.1 --- Preparation of SDS-polyacrylamide gel --- p.39 / Chapter 2.13.2 --- Preparation of protein samples --- p.39 / Chapter 2.13.3 --- SDS-polyacrylamide gel electrophoresis --- p.40 / Chapter 2.13.4 --- Protein transfer to nylon membrane --- p.40 / Chapter 2.13.5 --- Antibodies and detection --- p.40 / Chapter 2.13.6 --- Stripping membrane --- p.41 / Chapter 2.14 --- Bromodeoxyuridine proliferation assay --- p.42 / Chapter 2.14.1 --- Bromodeoxyuridine labeling and detection --- p.42 / Chapter 2.14.2 --- Cell number determination --- p.42 / Chapter 2.15 --- Fluorescence microscopy --- p.43 / Chapter 2.16 --- Confocal microscopy --- p.43 / Chapter 2.17 --- Statistical analysis --- p.44 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Sequence analysis --- p.45 / Chapter 3.1.1 --- Primary structure analysis --- p.45 / Chapter 3.1.2 --- Transmembrane he lice prediction --- p.46 / Chapter 3.1.3 --- Conserved domain prediction --- p.51 / Chapter 3.1.4 --- Sequence alignments across different species --- p.52 / Chapter 3.2 --- Differential expression during rat myocardial development --- p.53 / Chapter 3.3 --- Altered mRNA levels during differentiation of H9C2 cells --- p.55 / Chapter 3.4 --- "Cloning of IFITMl, IFITM2 and IFITM3" --- p.60 / Chapter 3.5 --- Subcellular localization --- p.61 / Chapter 3.5.1 --- Fluorescence microscopy --- p.61 / Chapter 3.5.2 --- Subcellular fractionation --- p.70 / Chapter 3.6 --- "In vitro induction by interferons-α, β and γ" --- p.72 / Chapter 3.7 --- "DNA synthesis after in vitro induction of interferons-α, β and γ" --- p.79 / Chapter 3.8 --- "Proliferating cell nuclear antigen expression after in vitro induction of interferons-α, β and γ" --- p.87 / Chapter 3.9 --- "DNA synthesis after overexpression of IFITM1, IFITM2 and IFITM3" --- p.93 / Chapter 3.10 --- "Proliferating cell nuclear antigen expression after overexpression of IFITM1, IFITM2 and IFITM3" --- p.95 / Chapter 3.11 --- "β-catenin and cyclin D1 expression after in vitro induction of interferons-α, β and γ" --- p.97 / Chapter 3.12 --- "β-catenin and cyclin D1 expression after overexpression of IFITMl, IFITM2 and IFITM3" --- p.101 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- "Upregulation of IlFITMl, IFITM2 and IFITM3 during myocardial development" --- p.103 / Chapter 4.2 --- "Subcellular localization of IFITMl, IFITM2 and IFITM3" --- p.105 / Chapter 4.3 --- "Induction by interferons-α, β and γ" --- p.107 / Chapter 4.4 --- Inhibition of DNA synthesis by interferons-α and β and IFITM1 --- p.109 / Chapter 4.5 --- Involvement of IFITM family in canonical Wnt pathway --- p.112 / Chapter 4.6 --- Other possible pathways involved --- p.117 / Chapter CHAPTER 5 --- FUTURE PROSPECTS / Chapter 5.1 --- Production of antibodies --- p.118 / Chapter 5.2 --- Silencing or knockout approach --- p.118 / Chapter 5.3 --- Target genes of Wnt/β-catenin signaling --- p.119 / Chapter 5.4 --- Other signaling pathways involved --- p.119 / Chapter 5.5 --- Use of primary cardiomyocytes --- p.120 / APPENDIX --- p.121 / REFERENCES --- p.124

Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures.   The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z. / QC 20110608

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Proteins, which regulate most of the biological activities, perform their functions through their unique three-dimensional structures. The folding process of this three dimensional structure from one dimensional sequence is not well understood. The available facts infer that the protein structures are mostly conserved while sequences are more tolerant to mutations i.e. a number of sequences can adopt the same fold. These arch of optimal sequences for a chosen conformation is known as inverse protein folding and this thesis takes this approach to solve the enigmatic problem. This thesis presents a protein sequence design method based on the native state topology of protein structure. The structural importance of the amino acid positions has been converted into the topological parameter of the protein conformation. This scheme of extraction of topology of structures has been successfully applied on three dimensional lattice structures and in turn sequences with minimum energy for a given structure are obtained. This technique along with the reduced amino cid alphabet(A reduced amino acid alphabet is any clustering of twenty amino acids based on some measure of the irrelative similarity) has been applied on the protein structures and hence designed optimal amino acid sequences for a given structure. These designed sequences are energetically much better than the native amino acid sequence. The utility of this method is further confirmed by showing the similarity between naturally occurring and the designed sequences. In summary, a computationally efficient method of designing optimal sequences for a given structure is given. The physical interaction energy between the amino acids is an important part of study of protein-protein interaction, structure prediction, modeling and docking etc. The local environment of amino acids makes a difference between the same amino acid pairs in the protein structure and so the pair-wise interaction energy of amino acid residues should depend on the irrespective environment. A local environment depended knowledge based potential energy function is developed in this thesis. Two different environments, one of these is the local degree (number of contacts) and the other is the secondary structural element of amino acids, have been considered. The investigations have shown that the environment-based interaction preferences for amino acids is able to provide good potential energy functions which perform exceedingly well in discriminating the native structure from the structures with random interactions. Further, the membrane proteins are located in a completely different physico-chemical environment with different amino acid composition than the water soluble proteins. This work provides reliable potential energy functions which take care of different environment for the investigation(model/predict) of the structure of helical membrane proteins. Three different environments, parallel and perpendicular to the lipid bilayer and number of amino acid contacts, are explored to analyze the environmental effects on the potential functions. These environment dependent scoring functions perform exceedingly well indiscriminating the native sequence from a set of random sequences. Hydrophobicity of amino acids is a measure of buriedness or exposure to the aqueous environment. The lack of uniformity within the protein environment gives rise to the different values of hydrophobicity for the same amino acids, which completely depends on its location inside the protein.The contact based environment dependent hydrophobicity values of all amino acids, separately for globular and membrane proteins, have also been evaluated in this thesis. Apart from developing scoring functions, the packing of helices in membrane proteins is investigated by an approach based on the local backbone geometry and side chain atom-atom contacts of amino acids. A parameter defined in this study is able to capture the essential features of inter-helical packing, which may prove to be useful in modeling of helical membrane proteins. In conclusion, this thesis has described a novel technique to design the energetically minimized amino acid sequences which can fold in to a given conformation. Also the environment dependent interaction preference of amino acids in globular proteins is captured an efficient manner. Specially, the environment dependent scoring function for helical membrane proteins is a first successful attempt in this direction.

Membrane proteins roughly constitute 30% of open reading frames in a genome and form 70% of current drug targets. They are classified as integral, peripheral membrane proteins and polypeptide toxins. α-helices and β -strands are the principal secondary structures observed in integral membrane proteins. This thesis presents the results of studies on analysis and correlation of sequence and structure of helices constituting integral helical membrane proteins. The aim of this work is to understand the helix stabilization, distortion as well as packing in terms of amino acid sequences and the correlated structures they adopt. To this end, analyses of datasets of X-ray crystal structures of integral helical membrane proteins and their comparison with a dataset of representative folds of globular proteins was carried out. Initial analysis was carried out using a non-redundant dataset of 75 membrane proteins to understand sequence and structural preferences for stabilization of helix termini. The subsequent analysis of helix distortions in membrane proteins was carried out using an updated dataset of 90 membrane proteins. Chapter 1 of the thesis reviews experimental as well as theoretical studies that have provided insights into understanding the structure of helical membrane proteins. Chapter 2 details the methods used during the course of the present investigations. These include the protocol used for creation of the non-redundant database of membrane and globular proteins. Various statistical methods used to test significance of the position-wise representation of amino acids in helical regions and the differences in globular and membrane protein datasets have been listed. Based on the tests of significance, a methodology to identify differences in propensity values that are statistically significant among two datasets has been devised. Programs used for secondary structure identification of membrane proteins namely Structure Identification (STRIDE) and Assignment of Secondary Structure in Proteins (ASSP) as well as those used for characterization of helical geometry (Helanal-Plus) have also been enlisted. In Chapter 3, datasets of 865 α-helices in 75 membrane proteins and 2680 α- helices from 626 representative folds in globular proteins defined by the STRIDE program have been analyzed to study the sequence determinants at fifteen positions within and around the α-helix. The amino acid propensities have been studied for positions that are important for the process of helix initiation, propagation, stabilization and termination. Each of the 15 positions has unique sequence characteristics reflecting their role and contribution towards the stability of the α-helix. A comparison of the sequence preferences in membrane and globular proteins revealed common residue preferences in both these datasets confirming the importance of these positions and the strict residue preferences therein. However, short/medium length α-helices that initiated/terminated within the membrane showed distinct amino acid preferences at the N-terminus (Ncap, N1, N2) as well as the C-terminus ( Ccap, Ct) when compared to α-helices belonging to membrane and globular proteins. The sequence preferences in membrane proteins were governed by the helix initiating and terminating property of the amino acids as well as the external environment of the helix. Results from our analysis also conformed well with experimentally tested amino acid preferences in a position-specific amino acid preference library of the rat neurotensin receptor (Schlinkmann et al (2012) Proc Natl Acad Sci USA 109(25):1890-5) as well as crystal structures of GPCR proteins. In the light of the environment dependent amino acid preferences found at α- helix termini, a survey was carried out to find various helix capping motifs adopted at both termini of α-helices in globular and membrane proteins to stabilize these helix termini. The results from these findings have been reported in Chapter 4. A sequence dependent structural preference is found for capping motifs at helix termini embedded inside and protruding outside the membrane. The N-terminus of α-helices was capped by hydrogen bonds involving free main chain amide groups of the first helical turn as donors and amino acid side chains as acceptors, as against the C-terminus which showed position-dependent characteristic backbone conformations to cap the helix. Overall helix termini inside the membrane did not show a very high number of capping motifs; instead these termini were stabilized by helix- helix interactions contributed by the neighboring helices of the helical bundle. In Chapter 5, we examine transmembrane helical (TMH) regions to identify as well as characterize the various types of helix perturbations in membrane proteins using ASSP and Helanal-Plus. A survey of literature shows that the term ‘helix kink’ has been used rather loosely when in fact helical regions show significant amounts of variation and transitions in helical parameters. Hence a systematic analysis of TMH regions was undertaken to quantify different types of helix perturbations, based on geometric parameters such as helical twist, rise per residue and local bending angle. Results from this analysis indicated that helices are not only kinked but undergo transitions to form interspersed stretches of 310 helices and π-bulges within the bilayer. These interspersed 310 and π-helices showed unique sequence preferences within and around their helical body, and also assisted in main- taining the helical structure within the bilayer. We found that Proline not only kinked the helical regions in a characteristic manner but also caused a tightening or unwinding in a helical region to form 310 and π-helix fragments respectively. The helix distortions also resulted in backbone hydrogen bonds to be missed which were stabilized by hydrogen bonds from neighboring residues mediated by their side chain atoms. Furthermore, a packing analysis showed that helical regions with distortions were able to establish inter-helical interactions with more number of transmembrane segments in the helical bundle. The study on helix perturbations presented in the previous chapter, brought to light a previously unreported 19 amino acid π-helix fragment interspersed between α-helices in the functionally important transmembrane helix 2 (TM2) belonging to Mitochondrial cytochrome-c-oxidase (1v55). Chapter 6 describes a case study of the structurally similar but functionally different members within the Heme-Copper- Superoxidases (HCO) superfamily that were considered for a comparative analysis of TM2. An analysis of 7 family members revealed that the π-helix shortens, fragments in two shorter π-helices or was even absent in some family members. The long π-helix significantly decreased the total twist and rise of the entire helical fragment thus accommodating more hydrophobic amino acids within the bilayer to avoid hydrophobic mismatch with the bilayer. The increased radius of the TM2 helical fragment also assisted in helix packing interactions by increasing the number of residues involved in helix-helix interactions and hydrogen bonds. Chapter 7 documents the conclusions from the different analyses presented in each of the above chapters. Overall, it is found that membrane proteins optimize the biophysical and chemical constraints of the external environment to strategically place select amino acids at helix termini to ‘start’ and ‘stop’ α-helices. The stabilization of these helix termini is a consequence of sequence dependent structural preferences to form helix capping motifs. The studies on helix transitions and distortions highlight that membrane proteins are not only packed as α-helices but also accomodate 310- and π-helical fragments. These transitions and distortions help in harboring more hydrophobic amino acids and aiding inter-helical interactions important for maintaining the fold of the membrane protein. Appendix A describes a comparison of α-helix assignments in globular and membrane proteins by two algorithms, one based on Cα trace (ASSP) and the other using a combination of hydrogen bond pattern along with backbone torsion angles φ and ψ (STRIDE).

Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.

<p dir="ltr">The obligate human pathogen, <i>Neisseria gonorrhoeae </i>(Ngo), has continued to acquire widespread antibiotic resistance. Ngo is the causative agent of the sexually transmitted disease gonorrhea, and can cause additional complications such as endocarditis, septicemia, and infertility if left untreated. The Centers for Disease Control and Prevention (CDC) now recommends a treatment option of a single drug of last resort, ceftriaxone, leaving a need for novel therapeutics against this pathogen.</p><p dir="ltr">Like many bacterial pathogens, Ngo is Gram-negative consisting of both an inner membrane (IM) and outer membrane (OM). The transmembrane proteins in the IM have primarily an α-helical fold, while the transmembrane proteins in the OM have a β-barrel fold. These β-barrel outer membrane proteins (OMPs) have essential functions in regulating the homeostasis and nutrient acquisition of the cell, in addition to promoting virulence in pathogenic strains. These OMPs are folded and inserted into the outer membrane by the β-barrel assembly machinery (BAM) complex. In <i>E. coli,</i> BAM consists of five proteins: BamA, an OMP itself, and four lipoproteins, BamB, C, D, and E.</p><p dir="ltr">Here we present our work toward the structural characterization of BAM from Ngo (<i>Ng</i>BAM) using cryo-EM. Ngo lack a homolog of BamB and may function as a four component complex. To better understand the mechanism for how <i>Ng</i>BAM is able to mediate OMP biogenesis despite lacking a component that is critical in <i>E. coli</i>, we determined the cryo-EM structure of <i>Ng</i>BAM, which revealed several distinct features including that the barrel domain of BamA being observed in the inward-open conformation. We also investigated <i>Ng</i>BAM as a therapeutic target, by studying its interaction with a novel broad spectrum antibiotic darobactin. We first showed darobactin is effective against the laboratory strains of NgoFA19 and ATCC-49226. We also show it is effective against the human isolate WHOX, with a comparable MIC to ceftriaxone. To structurally characterize the mechanism of inhibition by darobactin, we used cryo-EM to determine the structures of <i>Ng</i>BAM bound to two darobactin compounds. In these structures, darobactin binding was accompanied by large conformational changes in <i>Ng</i>BamA. To further probe the effects of darobactin on the conformational plasticity of <i>Ng</i>BAM we performed experiments using double electron-electron resonance spectroscopy, which showed distance changes between the engineered site labels consistent with the conformational changes observed in our structural observation. In addition, narrowing of the peak distributions indicated that darobactin binding was reducing the overall conformational heterogeneity of the complex. Taken together, the work presented here contributes to the understanding of how <i>Ng</i>BAM functions in folding and inserting OMPs and provides a foundation for future structure based drug design of darobactin and other potential compounds.</p>