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Characterization of Membrane Proteins: from a Gated Plant Aquaporin to Animal Ion Channel Receptors Characterization of Membrane Proteins: From a gated plant aquaporin to animal ion channel receptors Survery, Sabeen 2015 Link to publication Citation for published version (APA): Survery, S. (2015). Characterization of Membrane Proteins: From a gated plant aquaporin to animal ion channel receptors. Department of Biochemistry and Structural Biology, Lund University. 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LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 Printed by Media-Tryck, Lund University 2015 SURVERY SABEEN Characterization of Membrane Proteins - From a gated plant aquaporin to animal ion channel receptors channel ion animal to aquaporin plant gated a From - Proteins Membrane of Characterization Characterization of Membrane Proteins From a gated plant aquaporin to animal ion channel receptors SABEEN SURVERY | FACULTY OF SCIENCE | LUND UNIVERSITY 2015 LUND UNIVERSITY Faculty of Science Department of Biochemistry and Structural Biology 224030 Center for Molecular Protein Science ISBN 978-91-7422-403-0 789174 9 Characterization of Membrane Proteins From a gated plant aquaporin to animal ion channel receptors Sabeen Survery DOCTORAL DISSERTATION Academic thesis which, by due permission of the Faculty of Science, Lund University, Sweden, will be defended on Friday 12 June, 2015, at 1015 in lecture hall B at the Centre of Chemistry and Chemical Engineering, Getingevagen 60, Lund. Faculty opponent: Professor Dr. Eric Beitz, Pharmaceutical and Medicinal Chemistry University of Kiel, Germany 1 Organization: Document name: Doctoral dissertation Lund University Biochemistry and Structural Biology Center for Molecular Protein Science, PO Box 124, 221 00 Lund, Sweden Date of issue Author: Sabeen Survery Sponsoring organization Title and subtitle: Characterization of Membrane Proteins: from a gated aquaporin to animal ion channel receptors Abstract Membrane proteins play several important roles in a cell. Among these proteins are aquaporins (AQPs) and transient receptor potential (TRP) ion channels that mediate water transport, temperature and noxious chemical sensation, respectively. The function of some AQPs, for example the spinach isoform SoPIP2;1 is regulated by pH, phosphorylation and heavy metals such as mercury. However, the mechanisms by which mercury activate or inhibits AQPs are poorly understood. We suggest that mercury binds to SoPIP2;1 close to the C-terminal end and that the binding of mercury results in destabilization of the C-terminal region. This may affect its interaction with the residues forming the gate and therefore lead to an increase of the water permeability of SoPIP2;1 (Paper II). SoPIP2;1 is a highly selective water channel and can be produced as a functional protein in high yield in a heterologous system which suggest that SoPIP2;1 is a good choice for insertion in biomimetic membranes to be used for water purification. However, the stability of SoPIP2;1 in artificial membranes needed to be demonstrated. Thus we determined the stability of SoPIP2;1 in different lipids and identified E. coli polar lipids as the best system for reconstitution of SoPIP2;1. The results will contribute towards the effort to use SoPIP2;1 in biomimetic water filtration technology (Paper I). The animal TRP ion channel subtype A1 (TRPA1) from fruit fly, snake and mosquito has been implicated in warm temperature sensation. However, the threshold temperature which activates human TRPA1 (hTRPA1) is controversial. We addressed this issue by reconstituting the purified hTRPA1 in artificial lipid membranes. The purified hTRPA1 was found to be activated by cold temperatures and electrophilic chemicals. The results resolve the controversy surrounding the threshold temperature for the activation of hTRPA1 (Paper IV). The Anopheles gambiae TRPA1 (AgTRPA1) was found to be activated by heat and electrophilic compounds when reconstituted in artificial membranes after purification. The temperature activation as well as the binding of electrophilic ligands to AgTRPA1 resulted in the quenching of fluorescence suggesting that thermal and chemical activation brought about similar conformational changes of the protein and perhaps reflect the dynamic change in the conformation of residues involved in the gating process (Paper III). We also demonstrated that the N-terminal domain of both human and mosquito TRPA1 is not essential for thermal/chemical sensation (Paper III and Paper IV) as opposed to previous reports. Key words: MIPs, AQPs, TRP ion channels, water transport, electrophilic compounds, themo sensor. Classification system and/or index terms (if any) Supplementary bibliographical information Language: English ISSN and key title ISBN 978-91-7422-403-0 Recipient’s notes Number of pages Price Security classification I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sources permission to publish and disseminate the abstract of the above- mentioned dissertation. Signature Date 2 Characterization of Membrane Proteins From a gated plant aquaporin to animal ion channel receptors Sabeen Survery 3 Back cover Malaria mosquito (Anopheles) photo credit to Centers for disease control and prevention (CDC). Copyright Sabeen Survery Faculty of Science Department of Biochemistry and Structural Biology ISBN 978-91-7422-403-0 Printed in Sweden by Media-Tryck, Lund University Lund 2015 4 To My Parents 5 6 Abstract Membrane proteins play several important roles in a cell. Among these proteins are aquaporins (AQPs) and transient receptor potential (TRP) ion channels that mediate water transport, temperature and noxious chemical sensation, respectively. The function of some AQPs, for example the spinach isoform SoPIP2;1 is regulated by pH, phosphorylation and heavy metals such as mercury. However, the mechanisms by which mercury activate or inhibits AQPs are poorly understood. We suggest that mercury binds to SoPIP2;1 close to the C-terminal end and that the binding of mercury results in destabilization of the C-terminal region. This may affect its interaction with the residues forming the gate and therefore lead to an increase of the water permeability of SoPIP2;1 (Paper II). SoPIP2;1 is a highly selective water channel and can be produced as a functional protein in high yield in a heterologous system which suggest that SoPIP2;1 is a good choice for insertion in biomimetic membranes to be used for water purification. However, the stability of SoPIP2;1 in artificial membranes needed to be demonstrated. Thus we determined the stability of SoPIP2;1 in different lipids and identified E. coli polar lipids as the best system for reconstitution of SoPIP2;1. The results will contribute towards the effort to use SoPIP2;1 in biomimetic water filtration technology (Paper I). The animal TRP ion channel subtype A1 (TRPA1) from fruit fly, snake and mosquito has been implicated in warm temperature sensation. However, the threshold temperature which activates human TRPA1 (hTRPA1) is controversial. We addressed this issue by reconstituting the purified hTRPA1 in artificial lipid membranes. The purified hTRPA1 was found to be activated by cold temperatures and electrophilic chemicals. The results resolve the controversy surrounding the threshold temperature for the activation of hTRPA1 (Paper IV). The Anopheles gambiae TRPA1 (AgTRPA1) was found to be activated by heat and electrophilic compounds when reconstituted in artificial membranes after purification. The temperature activation as well as the binding of electrophilic ligands to AgTRPA1 resulted in the quenching of fluorescence suggesting that thermal and chemical activation brought about similar conformational changes of the protein and perhaps reflect the dynamic change in the conformation of residues involved in the gating process (Paper III). We also demonstrated that the N- terminal domain of both human and mosquito TRPA1 is not essential for thermal/chemical sensation (Paper III and Paper IV) as opposed to previous reports. 7 8 The thesis is based on following papers I. Structure and stability of the Spinach Aquaporin SoPIP2;1 in detergent micelles and lipid membranes Plasencia I, Survery S, Ibragimova S, Hansen JS, Kjellbom P, Helix-Nielsen C, Johanson U, Mouritsen OG PLoS ONE. 2011 | Volume 6 | Issue 2 | SS took part in the protein expression and purification, performed some of the CD experiment, performed electrophoresis of temperature denatured protein samples and participated in writing of related parts of the paper. II. Increased permeability of SoPIP2;1 by mercury and mutations of cysteine in
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