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N- and C-terminal domains in tobacco aquaporins - Analysis of protein-mediated water permeability in vitro and in silico

Glitsos, Gabriel (2017)
N- and C-terminal domains in tobacco aquaporins - Analysis of protein-mediated water permeability in vitro and in silico.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

Aquaporins are a subclass of a ubiquitous protein family, the major intrinsic proteins (MIPs), and are thus represented in all domains of life. Their primordial function as integral membrane channels is the passive mediation of water across lipid bilayer barriers. In addition, various alternative substrates, such as small uncharged molecules, gases, carbohydrates, metalloids or ions have been found to be transported via aquaporins. As such, they fulfill a wide range of physiological functions and are of growing interest as targets for medical, as well as industrial applications. In plants, aquaporins are divided into five subclasses based on their localization, substrate specificity and sequence similarity: Plasma membrane intrinsic proteins (PIPs), tonoplast membrane intrinsic proteins (TIPs), Nodulin26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and uncategorized (X) intrinsic proteins (XIPs). PIPs as the largest group are further split up into PIP1 and PIP2 phylogenetic subcategories. The latter is differentiated from the former by a shorter N- and a longer C-terminus, an additional number of amino acids in the first extracellular loop A and a significantly higher overall water permeability. Furthermore, PIP2s have been described as rather strict water channels, whereas members of the PIP1 family are more likely to mediate alternative substrates. As typical representatives of their respective PIP subclasses, NtAQP1 and NtPIP2;1 from tobacco were at the center of this thesis. A detailed in silico amino acid sequence analysis and comparison revealed the most significant variances in terms of domain length and sequence identity to be in the N- and C-termini, as well as loop A of these two aquaporins. A previous study found NtAQP1 water permeation to be unmodulated after its loop A was modified to resemble that of a PIP2 member. In addition, a multiple sequence alignment with various other MIPs helped identify all sequence motifs relevant for substrate specificity in NtAQP1 and NtPIP2;1. Interestingly, all of them were found to be identical between the two, thus giving way to the hypothesis that their terminal domains could play a significant role in their respective water permeation capabilities. In order to test that hypothesis, an E. coli based continuous exchange cell free expression (CECF) system was established. A total of three different expression modes were tested for experimental applicability. The precipitation based mode (P-CF) without the inclusion of a hydrophobic environment served as a quick initial expression test for newly constructed vectors, as well as verification of individual reaction components. Detergent based cell free expression (D-CF) provided micelles for the direct solubilization of translated aquaporin, but was eventually dismissed as a viable option due to the complexity of its downstream processing. Finally, lipid based cell free expression (L-CF) provided both a liposome based hydrophobic environment for direct integration of translated protein and a downstream processing of comparably low complexity. The water permeability of proteoliposomes containing either of the two tobacco aquaporin wildtypes or terminal domain mutant constructs, as well as empty control liposomes was measured via a Stopped Flow based assay. Therein, (proteo)liposome shrinkage via a hypoosmotic pressure gradient was analyzed via scattered light kinetics. Obtained raw data underwent nonlinear regression to an exponential rise function and thus allowed the calculation of the water permeability factor Pf for individual samples. NtPIP2;1 and NtAQP1 showed water permeation rates in line with previously published results. While the former was a true aquaporin and demonstrated high water permeability, the latter indicated low transport rates barely above that of empty control liposomes. The deletion of either the N- or C-terminal domain in NtPIP2;1 caused a significant drop in water permeability to levels equivalent to NtAQP1 and the control (N) or slightly above that (C). Compared to that, the double deletion mutant demonstrated even lower water transport rates. In contrast, the removal of either of the NtAQP1 termini did not cause a significant shift in water permeation compared to the wildtype configuration. Deleting both domains, however, resulted in the lowest measured water permeability of all tested constructs. Subsequently, terminal domains were exchanged between the two wildtypes and the impact on their mediation functionality was analyzed. Pf values for NtPIP2;1 constructs, where either of the termini were exchanged with NtAQP1 sequences did not differ significantly from their single deletion counterparts. However, the exchange of both PIP2 termini resulted in transport rates statistically equivalent to those of the wildtype NtAQP1 and thus significantly higher than NtPIP2;1 with both domains removed. Finally, all NtAQP1 terminal exchange mutants demonstrated increased water permeation in the following order, when compared with the wildtype: N exchange < C exchange < N & C exchange, with the latter closing two thirds of the water transport gap previously seen between the two wildtype aquaporins. Thus, all three NtAQP1 exchange mutants showed significantly higher transport rates than their deletion mutant equivalents. Based on the obtained results, various types of previously reported aquaporin regulation were discussed in order to better interpret the role of terminal domains in the water permeability of NtPIP2;1 and NtAQP1. N-terminal acetylation / methylation as a type of post-translational modification, the interaction of termini with neighboring aquaporin monomers and a modification of mechanosensitivity were found to be options in the realm of possibility. In addition, a three-dimensional structure homology modeling of both wildtypes, as well as their respective double deletion and double exchange mutants allowed to spot potential conformation changes in transmembrane regions. In conclusion, both the mere presence, as well as the specific sequence makeup of the terminal domains seem to play a major role in the water transport functionality of both tobacco aquaporins.

Typ des Eintrags: Dissertation
Erschienen: 2017
Autor(en): Glitsos, Gabriel
Art des Eintrags: Erstveröffentlichung
Titel: N- and C-terminal domains in tobacco aquaporins - Analysis of protein-mediated water permeability in vitro and in silico
Sprache: Englisch
Referenten: Kaldenhoff, Prof. Dr. Ralf ; Warzecha, Prof. Dr. Heribert
Publikationsjahr: April 2017
Ort: Darmstadt
Datum der mündlichen Prüfung: 10 März 2017
URL / URN: http://tuprints.ulb.tu-darmstadt.de/6124
Kurzbeschreibung (Abstract):

Aquaporins are a subclass of a ubiquitous protein family, the major intrinsic proteins (MIPs), and are thus represented in all domains of life. Their primordial function as integral membrane channels is the passive mediation of water across lipid bilayer barriers. In addition, various alternative substrates, such as small uncharged molecules, gases, carbohydrates, metalloids or ions have been found to be transported via aquaporins. As such, they fulfill a wide range of physiological functions and are of growing interest as targets for medical, as well as industrial applications. In plants, aquaporins are divided into five subclasses based on their localization, substrate specificity and sequence similarity: Plasma membrane intrinsic proteins (PIPs), tonoplast membrane intrinsic proteins (TIPs), Nodulin26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and uncategorized (X) intrinsic proteins (XIPs). PIPs as the largest group are further split up into PIP1 and PIP2 phylogenetic subcategories. The latter is differentiated from the former by a shorter N- and a longer C-terminus, an additional number of amino acids in the first extracellular loop A and a significantly higher overall water permeability. Furthermore, PIP2s have been described as rather strict water channels, whereas members of the PIP1 family are more likely to mediate alternative substrates. As typical representatives of their respective PIP subclasses, NtAQP1 and NtPIP2;1 from tobacco were at the center of this thesis. A detailed in silico amino acid sequence analysis and comparison revealed the most significant variances in terms of domain length and sequence identity to be in the N- and C-termini, as well as loop A of these two aquaporins. A previous study found NtAQP1 water permeation to be unmodulated after its loop A was modified to resemble that of a PIP2 member. In addition, a multiple sequence alignment with various other MIPs helped identify all sequence motifs relevant for substrate specificity in NtAQP1 and NtPIP2;1. Interestingly, all of them were found to be identical between the two, thus giving way to the hypothesis that their terminal domains could play a significant role in their respective water permeation capabilities. In order to test that hypothesis, an E. coli based continuous exchange cell free expression (CECF) system was established. A total of three different expression modes were tested for experimental applicability. The precipitation based mode (P-CF) without the inclusion of a hydrophobic environment served as a quick initial expression test for newly constructed vectors, as well as verification of individual reaction components. Detergent based cell free expression (D-CF) provided micelles for the direct solubilization of translated aquaporin, but was eventually dismissed as a viable option due to the complexity of its downstream processing. Finally, lipid based cell free expression (L-CF) provided both a liposome based hydrophobic environment for direct integration of translated protein and a downstream processing of comparably low complexity. The water permeability of proteoliposomes containing either of the two tobacco aquaporin wildtypes or terminal domain mutant constructs, as well as empty control liposomes was measured via a Stopped Flow based assay. Therein, (proteo)liposome shrinkage via a hypoosmotic pressure gradient was analyzed via scattered light kinetics. Obtained raw data underwent nonlinear regression to an exponential rise function and thus allowed the calculation of the water permeability factor Pf for individual samples. NtPIP2;1 and NtAQP1 showed water permeation rates in line with previously published results. While the former was a true aquaporin and demonstrated high water permeability, the latter indicated low transport rates barely above that of empty control liposomes. The deletion of either the N- or C-terminal domain in NtPIP2;1 caused a significant drop in water permeability to levels equivalent to NtAQP1 and the control (N) or slightly above that (C). Compared to that, the double deletion mutant demonstrated even lower water transport rates. In contrast, the removal of either of the NtAQP1 termini did not cause a significant shift in water permeation compared to the wildtype configuration. Deleting both domains, however, resulted in the lowest measured water permeability of all tested constructs. Subsequently, terminal domains were exchanged between the two wildtypes and the impact on their mediation functionality was analyzed. Pf values for NtPIP2;1 constructs, where either of the termini were exchanged with NtAQP1 sequences did not differ significantly from their single deletion counterparts. However, the exchange of both PIP2 termini resulted in transport rates statistically equivalent to those of the wildtype NtAQP1 and thus significantly higher than NtPIP2;1 with both domains removed. Finally, all NtAQP1 terminal exchange mutants demonstrated increased water permeation in the following order, when compared with the wildtype: N exchange < C exchange < N & C exchange, with the latter closing two thirds of the water transport gap previously seen between the two wildtype aquaporins. Thus, all three NtAQP1 exchange mutants showed significantly higher transport rates than their deletion mutant equivalents. Based on the obtained results, various types of previously reported aquaporin regulation were discussed in order to better interpret the role of terminal domains in the water permeability of NtPIP2;1 and NtAQP1. N-terminal acetylation / methylation as a type of post-translational modification, the interaction of termini with neighboring aquaporin monomers and a modification of mechanosensitivity were found to be options in the realm of possibility. In addition, a three-dimensional structure homology modeling of both wildtypes, as well as their respective double deletion and double exchange mutants allowed to spot potential conformation changes in transmembrane regions. In conclusion, both the mere presence, as well as the specific sequence makeup of the terminal domains seem to play a major role in the water transport functionality of both tobacco aquaporins.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Aquaporine sind eine Unterklasse einer ubiquitären Proteinfamilie, den major intrinsic proteins (MIPs), und sind als solche in allen Domänen des Lebens vertreten. Ihre ursprüngliche Funktion als integrale Membranproteine ist der passive Transport von Wasser durch Doppellipidmembranen. Darüber hinaus wurden weitere Alternativen wie kleine, ungeladene Moleküle, Gase, Kohlenhydrate, Metalloide oder Ionen als Substrate von Aquaporinen beschrieben. Dank dieses breiten Spektrums erfüllen die Membrankanäle eine große Bandbreite von physiologischen Funktionen und rücken vermehrt in den wissenschaftlichen Fokus als Targets für pharmakologische und industrielle Applikationen. In Pflanzen wurde eine Aufteilung von Aquaporinen in insgesamt fünf Unterklassen durchgeführt, welche auf Lokalisation, Substratspezifität und Sequenzidentität basiert: plasma membrane intrinsic proteins (PIPs), tonoplast membrane intrinsic proteins (TIPs), Nodulin26-like intrinsic proteins (NIPs), small, basic intrinsic proteins (SIPs) und uncategorized (X) intrinsic proteins (XIPs). Die zahlenmäßig größte Gruppe der PIPs lässt sich weiter in die phylogenetischen Unterkategorien PIP1 und PIP2 einteilen. Letztere unterscheiden sich von Ersteren durch einen kürzeren N- und einen längeren C-terminus, zusätzlichen Aminosäuren in der ersten extrazellulären loop A und eine signifikant höhere intrinsische Wasserpermeabilität. Des Weiteren wurden PIP2 Aquaporine als strikte Wasserkanäle beschrieben während bei Mitgliedern der PIP1 Unterkategorie die Wahrscheinlichkeit höher ist, auch alternative Substrate zu vermitteln. Im Fokus dieser Arbeit standen NtAQP1 und NtPIP2;1 aus Nicotiana tabacum als typische Repräsentanten ihrer jeweiligen Unterkategorien. Eine detaillierte in silico Analyse der beiden Aminosäuresequenzen mit anschließendem Vergleich offenbarte die größten Unterschiede bezüglich Sequenzlänge und -identität bei den N- und C-terminalen Proteindomänen sowie loop A dieser Aquaporine. Eine vorangegangene Studie demonstrierte eine unveränderte Wasserpermeabilität von NtAQP1, nachdem die Aminosäuresequenz von loop A an die eines PIP2 Aquaporins angepasst worden war. Unter Zuhilfenahme eines multiple sequence alignments mit diversen anderen MIPs wurden in der vorliegenden Arbeit alle für die Substratspezifität relevanten Sequenzmotive in NtAQP1 und NtPIP2;1 identifiziert. Interessanterweise stellte sich im Vergleich hierbei heraus, dass diese in allen Positionen identisch sind. Dies führte zur Formulierung der zentralen Hypothese dieser Arbeit, welche den terminalen Domänen der beiden Tabak-Aquaporine eine wichtige Rolle in der Modulation ihrer respektiven Wasserpermeabilität zusprach. Um diese Hypothese zu testen, wurde ein E. coli basiertes continuous exchange cell free expression (CECF) System etabliert. Hierbei wurden drei unterschiedliche Expressions-Modi auf ihre experimentelle Anwendbarkeit überprüft. Ein Präzipitations-basierter Modus (P-CF) ohne hydrophobe Additive diente als Schnelltest, sowohl zur Ermittlung der generellen Expressionsviabilität von neu konstruierten Vektoren, als auch zur Verifizierung individueller Reaktionskomponenten. Detergenz-basierte zellfreie Expression (D-CF) stellte zwar Mizellen zur direkten Solubilisierung von translatiertem Aquaporin zur Verfügung, wurde letztendlich aufgrund der Komplexität des notwendigen downstream processing als unpraktikable Option aufgegeben. Schlussendlich bot ein Lipid-basierter Modus (L-CF) nicht nur ein Liposom-abhängiges, hydrophobes Mileu zur unmittelbaren Integration von produziertem Protein, sondern auch ein downstream processing von vergleichsweise niedriger Komplexität. Die Wasserpermeabilität von Proteoliposomen mit eingebautem Wildtyp-Aquaporin oder entsprechenden Terminaldomän-Mutanten einerseits und leeren Kontroll-Liposomen andererseits wurde mittels eines Stopped Flow basierten Assays ermittelt. (Proteo)liposom-Schrumpfung wurde hierbei durch einen angelegten hypoosmotischen Gradienten ausgelöst und als Streulicht gemessen. Aufgenommene Rohdaten unterliefen eine nonlineare Regressionsanalyse anhand einer Exponential-Funktion und erlaubten somit die Kalkulation des Wasserpermeabilitätsfaktor Pf für individuelle Proben. NtPIP2;1 und NtAQP1 demonstrierten Wasserpermeabilitätsraten übereinstimmend mit zuvor publizierten Daten. Während ersteres hohe intrinsischen Wassertransport zeigte, weiste letzteres sehr niedrige Permeabilität auf, welche nur geringfügig höher war als bei leeren Kontroll-Liposomen. Eine Deletion der N- oder C-terminalen Domäne führte bei NtPIP2;1 zu einem signifikanten Rückgang der Wasserpermeabilität, welche sich dann auf NtAQP1 Wildtyp-Level (N-Deletion) bzw. geringfügig darüber (C-Deletion) einpendelte. Im Vergleich dazu zeigte die Doppeldeletions-Mutante die geringsten Transportraten. Das Entfernen von einzelnen terminalen Domänen bei NtAQP1 hatte keine signifikante Änderung der Wasserpermeabilität zur Folge. Eine Deletion beider Domänen zeigte jedoch die niedrigsten Transportraten aller gemessenen Konstrukte. Im Anschluß wurden N- und C-terminale Domänen zwischen den beiden Wildtyp-Aquaporinen ausgetauscht und die Auswirkungen im Hinblick auf die Modulation der Wassertransportraten analysiert. Pf Werte von NtPIP2;1 Konstrukten, in denen eine von beiden terminalen Domänen mit NtAQP1 Sequenzen ausgetauscht wurde, unterschieden sich nicht signifikant von denen ihrer respektiven Einzeldeletionsmutanten. Allerdings resultierte der Austausch beider PIP2 Termini in Transportraten statistisch äquivalent zu Werten vom NtAQP1 Wildtyp und somit signifikant höher als der NtPIP2;1 Doppeldeletionsmutante. Alle terminalen Austauschmutanten von NtAQP1 zeigten höhere Wasserpermeabilität als der Wildtyp und ordneten sich in die folgende Reihenfolge ein: N-terminaler Austausch < C-terminaler Austausch < N- & C-terminaler Austausch. Letztere Mutante überbrückte damit zwei Drittel der ursprünglichen Permeabilitätslücke zwischen den Wildtypen. Folglich demonstrierten alle drei NtAQP1 Austauschmutanten signifikant höhere Transportraten als ihre Deletions-Äquivalente. Basierend auf den erhaltenen Egebnissen wurden verschiedene Arten von bereits publizierten Regulationsmechanismen bei Aquaporinen diskutiert, um den Einfluss von terminalen Domänen auf die Wasserpermeabilität bei NtPIP2;1 und NtAQP1 interpretieren zu können. N-terminale Acetylierung / Methylierung als post-translationale Modifikation, die Interaktion von Termini mit benachbarten Aquaporin-Monomeren und eine Modifikation der Mechanosensitivität wurden hierbei als mögliche Optionen gefunden. Des Weiteren diente ein durchgeführtes 3D homology modeling der beiden Wildtypen und ihrer respektiven Doppeldeletions- und Doppelaustauschmutanten als strukturelle Grundlage, um potenzielle Konformationsänderungen in Transmembranregionen nachzuweisen. Abschliessend lässt sich sagen, dass sowohl die bloße Anwesenheit, als auch spezifische terminale Sequenzen ausschlaggebend sind für die Modulation der Wassertransportfunktionalität in beiden Tabak-Aquaporinen.

Deutsch
URN: urn:nbn:de:tuda-tuprints-61247
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
500 Naturwissenschaften und Mathematik > 530 Physik
500 Naturwissenschaften und Mathematik > 540 Chemie
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
500 Naturwissenschaften und Mathematik > 580 Pflanzen (Botanik)
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie
10 Fachbereich Biologie > Applied Plant Sciences
Hinterlegungsdatum: 09 Apr 2017 19:55
Letzte Änderung: 09 Apr 2017 19:55
PPN:
Referenten: Kaldenhoff, Prof. Dr. Ralf ; Warzecha, Prof. Dr. Heribert
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 10 März 2017
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