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Polymer Supported Lipid Bilayer Membranes for the Integration of Transmembrane ProteinsRenner, Lars 04 May 2009 (has links) (PDF)
This work reports on the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) from natural occurring lipids as well as synthetic lipids on a set of polymer cushions consisting of alternating maleic acid copolymers. Maleic acid copolymers provide a versatile platform to adjust the physico-chemical behaviour by the choice of the comonomer unit. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solutions pH to 4. Upon formation the stability of sLBMs was not affected by subsequent variations of the environmental pH to 7.2. Even drastic changes in the environmental pH (between pH 2 and pH 9) did not lead to delamination and proved the stability of the polymer sLBM. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 µm2 s-1. An increase in cushion hydrophilicity correlated with a strong increase in the diffusion coefficient of the lipids. This trend was found to correlate with the kinetics of bilayer formation in the process of vesicle spreading. The observations strongly support the important role of the support’s polarity for the fluidity of the sLBM, which is probably related to the presence of a water layer between support and bilayer. The investigated polymer cushions are considered to open new options for the in situ modulation of lipid bilayer membranes characteristics to match the requirements for the successful integration of functional transmembrane proteins (TMPs). As each cushion exhibits different physico-chemical properties, the resulting behaviour of the sLBMs and TMPs could be exactly adjusted to the specific requirements of biological samples. This is exemplarily shown by the integration of the TMP beta amyloid precursor protein cleaving enzyme (BACE). Integrated BACE was observed to be mobile on all polymer cushions. On the contrary, no lateral mobility of BACE was found in solid sLBM. Furthermore, the activity of integrated BACE was analysed by the cleavage of an amyloid precursor protein analogue. Remarkably, the polymer cushions did not only enhance the mobility but were also found to increase the activity of BACE by a factor of 1.5 to 2.5 in comparison to solid sLBM. From the obtained results it is obvious that even small cytoplasmic domains of transmembrane proteins might not be preserved upon the integration in silica sLBM. The observed beneficial effects of the utilised polymer cushions on the mobility and activity of transmembrane proteins motivate further studies to clarify the general applicability of the polymer platform. Altogether, this polymer platform provides valuable options to form sLBM with varying characteristics to reconstitute transmembrane proteins for a wide range of possible future applications in biology. / Die vorliegende Arbeit beschreibt die Bildung von polymer unterstützten Lipiddoppelschichten zur Integration von transmembranen Proteinen. Das Polymerkissensystem besteht aus alternierenden Maleinsäurecopolymeren. Lipiddoppelschichten wurden durch die Steuerung der elektrostatischen Repulsion erzeugt: die Verringerung des pH-Wertes auf 4 wurde eine Erhöhung der adsorbierten Vesikelmenge auf den Polymeroberflächen induziert. Nach der erfolgten Bildung der Lipiddoppelschichten kann der pH-Wert beliebig variiert werden, ohne dass die Stabilität der Lipiddoppelschichten beeinflusst wird. Auch drastische Veränderungen des pH-Milieus (pH 2 - pH 9) führten zu keinen Veränderungen in der Membranintegrität. Der Grad der Hydrophilie und der Quellung der anionischen Polymerschichten beeinflusst sowohl die Bildung der Modellmembranen als auch die Mobilität der integrierten Lipidmoleküle. Dabei reichen die erzielten Lipiddiffusionskoeffizienten von 0.26 bis 2.6 µm2 s-1. Dabei ist die Mobilität direkt von der Hydrophilie des Substrates abhängig. Die beobachteten Ergebnisse zeigen deutlich die entscheidende Rolle der Polarität der verwendeten Substratoberflächen auf die Lipidmobilität, die sehr wahrscheinlich mit der Präsenz einer variablen Wasserschicht zusammenhängt. Die untersuchten Polymerkissen eröffnen neue Möglichkeiten für die insitu Modulierung der Charakteristika von Lipidschichten, um funktionale transmembrane Proteine zu integrieren. Aufgrund der unterschiedlichen physiko-chemischen Eigenschaften kann das Verhalten der Lipidschichten und der transmembranen Proteine nach den spezifischen Anforderungen des Modellsystems angepasst werden. Die funktionale Integration wurde am Beispiel des transmembranen Proteins BACE nachempfunden. Die Mobilität des integrierten BACE wurde auf allen Polymerkissen beobachtet. Im Gegensatz dazu wurde auf harten Substraten keine BACE Mobilität gefunden. Die Aktivität des integrierten BACE wurde durch die enzymatische Spaltung eines APP-Analogons nachgewiesen. Bemerkenswerteweise wurde ein Anstieg der BACE Aktivität auf den Polymerkissen um den Faktor 1,5 bis 2,5 im Vergleich zu den auf harten Substraten integrierten BACE beobachtet. Zusammenfassend, die verwendeten Polymerkissen bieten vielfältige Möglichkeiten Lipidschichten mit variierenden Eigenschaften für die Integration von transmembranen Proteinen zu erzeugen.
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Polymer Supported Lipid Bilayer Membranes for the Integration of Transmembrane ProteinsRenner, Lars 24 April 2009 (has links)
This work reports on the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) from natural occurring lipids as well as synthetic lipids on a set of polymer cushions consisting of alternating maleic acid copolymers. Maleic acid copolymers provide a versatile platform to adjust the physico-chemical behaviour by the choice of the comonomer unit. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solutions pH to 4. Upon formation the stability of sLBMs was not affected by subsequent variations of the environmental pH to 7.2. Even drastic changes in the environmental pH (between pH 2 and pH 9) did not lead to delamination and proved the stability of the polymer sLBM. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 µm2 s-1. An increase in cushion hydrophilicity correlated with a strong increase in the diffusion coefficient of the lipids. This trend was found to correlate with the kinetics of bilayer formation in the process of vesicle spreading. The observations strongly support the important role of the support’s polarity for the fluidity of the sLBM, which is probably related to the presence of a water layer between support and bilayer. The investigated polymer cushions are considered to open new options for the in situ modulation of lipid bilayer membranes characteristics to match the requirements for the successful integration of functional transmembrane proteins (TMPs). As each cushion exhibits different physico-chemical properties, the resulting behaviour of the sLBMs and TMPs could be exactly adjusted to the specific requirements of biological samples. This is exemplarily shown by the integration of the TMP beta amyloid precursor protein cleaving enzyme (BACE). Integrated BACE was observed to be mobile on all polymer cushions. On the contrary, no lateral mobility of BACE was found in solid sLBM. Furthermore, the activity of integrated BACE was analysed by the cleavage of an amyloid precursor protein analogue. Remarkably, the polymer cushions did not only enhance the mobility but were also found to increase the activity of BACE by a factor of 1.5 to 2.5 in comparison to solid sLBM. From the obtained results it is obvious that even small cytoplasmic domains of transmembrane proteins might not be preserved upon the integration in silica sLBM. The observed beneficial effects of the utilised polymer cushions on the mobility and activity of transmembrane proteins motivate further studies to clarify the general applicability of the polymer platform. Altogether, this polymer platform provides valuable options to form sLBM with varying characteristics to reconstitute transmembrane proteins for a wide range of possible future applications in biology. / Die vorliegende Arbeit beschreibt die Bildung von polymer unterstützten Lipiddoppelschichten zur Integration von transmembranen Proteinen. Das Polymerkissensystem besteht aus alternierenden Maleinsäurecopolymeren. Lipiddoppelschichten wurden durch die Steuerung der elektrostatischen Repulsion erzeugt: die Verringerung des pH-Wertes auf 4 wurde eine Erhöhung der adsorbierten Vesikelmenge auf den Polymeroberflächen induziert. Nach der erfolgten Bildung der Lipiddoppelschichten kann der pH-Wert beliebig variiert werden, ohne dass die Stabilität der Lipiddoppelschichten beeinflusst wird. Auch drastische Veränderungen des pH-Milieus (pH 2 - pH 9) führten zu keinen Veränderungen in der Membranintegrität. Der Grad der Hydrophilie und der Quellung der anionischen Polymerschichten beeinflusst sowohl die Bildung der Modellmembranen als auch die Mobilität der integrierten Lipidmoleküle. Dabei reichen die erzielten Lipiddiffusionskoeffizienten von 0.26 bis 2.6 µm2 s-1. Dabei ist die Mobilität direkt von der Hydrophilie des Substrates abhängig. Die beobachteten Ergebnisse zeigen deutlich die entscheidende Rolle der Polarität der verwendeten Substratoberflächen auf die Lipidmobilität, die sehr wahrscheinlich mit der Präsenz einer variablen Wasserschicht zusammenhängt. Die untersuchten Polymerkissen eröffnen neue Möglichkeiten für die insitu Modulierung der Charakteristika von Lipidschichten, um funktionale transmembrane Proteine zu integrieren. Aufgrund der unterschiedlichen physiko-chemischen Eigenschaften kann das Verhalten der Lipidschichten und der transmembranen Proteine nach den spezifischen Anforderungen des Modellsystems angepasst werden. Die funktionale Integration wurde am Beispiel des transmembranen Proteins BACE nachempfunden. Die Mobilität des integrierten BACE wurde auf allen Polymerkissen beobachtet. Im Gegensatz dazu wurde auf harten Substraten keine BACE Mobilität gefunden. Die Aktivität des integrierten BACE wurde durch die enzymatische Spaltung eines APP-Analogons nachgewiesen. Bemerkenswerteweise wurde ein Anstieg der BACE Aktivität auf den Polymerkissen um den Faktor 1,5 bis 2,5 im Vergleich zu den auf harten Substraten integrierten BACE beobachtet. Zusammenfassend, die verwendeten Polymerkissen bieten vielfältige Möglichkeiten Lipidschichten mit variierenden Eigenschaften für die Integration von transmembranen Proteinen zu erzeugen.
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Lipid Bilayers Supported by Multi-Stimuli Responsive PolymersKaufmann, Martin 25 March 2013 (has links) (PDF)
Artificial lipid bilayers formed on solid surface supports are widespread model systems to study physical, chemical, as well as biological aspects of cell membranes and fundamental interfacial interactions. The approach to use a thin polymer film representing a cushion for lipid bilayers prevents incorporated membrane proteins from pinning to the support and mimics the native environment of a lipid bilayer in certain aspects of the extracellular matrix and intracellular structures. A key component for cell anchorage to extracellular fibronectin is the transmembrane adhesion receptor alpha(5)beta(1) integrin. Its transport dynamics and clustering behavior plays a major role in the assembly of focal adhesions, which mediate mechanical forces and biochemical signals of cells with their surrounding.
The system investigated herein is envisioned to use extrinsically controlled stimuli-responsive polymer cushions to tune the frictional drag between polymer cushion and mobile membranes with incorporated integrins to actively regulate lipid membrane characteristics. To attain this goal, a temperature- and pH-responsive polymer based on poly(N-isopropylacrylamide) copolymers containing varying amounts of carboxyl-group-terminated comonomers at different aliphatic spacer lengths (PNIPAAm-co-carboxyAAM) was surface-grafted to a poly(glycidyl methacrylate) anchorage layer. The swelling transitions were characterized using atomic force microscopy, ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and found to be tunable over a wide range of temperature and pH. In agreement with the behavior of the polymers in solution, longer alkyl spacers decreased the phase transition temperature T(P) and higher contents of carboxylic acid terminated comonomers increased T(P) at alkaline conditions and decreased T(P) at acidic conditions. Remarkably, the point where the degree of carboxyl group deprotonation balances the T(P)-lowering effect of the alkyl spacer was distinctive for each alkyl spacer length.
These findings illustrate how the local and global balance of hydrophilic and hydrophobic interactions along the copolymer chain allows to adjust the swelling transition to temperatures below, comparable, or above those observed for PNIPAAm homopolymers. Additionally, it could be shown that surface-grafting leads to a decrease in T(P) for PNIPAAm homopolymers (7°C) and copolymers (5°C - 10°C). The main reason is the increase in local polymer concentration of the swollen film constrained by dense surface anchorage in comparison to the behavior of dilute free chains in solution. In accordance with the Flory-Huggins theory, T(P) decreases with increasing concentration up to the critical concentration. Biological functionalization of the PNIPAAm-co-carboxyAAm thin films was demonstrated for the cell adhesion ligand peptide cRGD via carbodiimide chemistry to mimic extracellular binding sites for the cell adhesion receptors integrin.
The outcome of QCM-D measurements of cRGD-functionalized surfaces showed a maintained stimuli-responsiveness with slight reduction in T(P). A drying/rehydration procedure of a 9:1 lipid mixture of the cationic lipid dioleoyl-trimethylammoniumpropane (DOTAP) and the zwitterionic dioleoyl-phosphatidylcholine (DOPC) was utilized to form lipid bilayer membranes on PNIPAAm-co-carboxyAAM cushions. Fluorescence recovery after photobleaching (FRAP) revealed that lipid mobility was distinctively higher (6.3 - 9.6) µm2 s-1 in comparison to solid glass support ((3.0 - 5.9) µm2 s-1). In contradiction to the initial expectations, modulation of temperature and pH led to poor variations in lipid mobility that did not correlate with the PNIPAAm cushion swelling state. The results suggested a weak coupling of the lipid bilayer with PNIPAAm polymer cushions that can be slightly tuned by electrostatic interactions.
The transmembrane adhesion receptor alpha(5)beta(1) integrin was reconstituted into liposomes consisting of DOPC/sphingomyelin/cholesterol 2:2:1 for the formation of polymer cushioned bilayers. PNIPAAm- co-carboxyAAM and maleic acid (MA) copolymers were used as cushions, both with the option for cRGD functionalization. On the MA copolymer cushions, fusion of proteoliposomes resulted in supported bilayers with mobile lipids as confirmed by FRAP. However, incorporated integrins were immobile. In an attempt to explain this observation, the medium-sized cytoplasmic integrin domain was accounted to hamper the movement by steric interactions with the underlying polymer chains in conjunction with electrostatic interactions of the cationic cytoplasmic domain with the oppositely charged MA copolymer.
On the PNIPAAm-co-carboxyAAM cushion only a drying/rehydration procedure lead to bilayer formation. However, again the integrins were immobile, presumably due to the harsh treatment during preparation. Nevertheless, the results of the investigated set of PNIPAAm copolymer films suggest their application as temperature- and pH-responsive switchable layers to control interfacial phenomena in bio-systems at different physiological conditions. The PNIPAAm-co-carboxyAAm cushioned bilayer system represents a promising step towards extrinsically controlled membrane – substrate interactions.
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Lipid Bilayers Supported by Multi-Stimuli Responsive PolymersKaufmann, Martin 08 February 2013 (has links)
Artificial lipid bilayers formed on solid surface supports are widespread model systems to study physical, chemical, as well as biological aspects of cell membranes and fundamental interfacial interactions. The approach to use a thin polymer film representing a cushion for lipid bilayers prevents incorporated membrane proteins from pinning to the support and mimics the native environment of a lipid bilayer in certain aspects of the extracellular matrix and intracellular structures. A key component for cell anchorage to extracellular fibronectin is the transmembrane adhesion receptor alpha(5)beta(1) integrin. Its transport dynamics and clustering behavior plays a major role in the assembly of focal adhesions, which mediate mechanical forces and biochemical signals of cells with their surrounding.
The system investigated herein is envisioned to use extrinsically controlled stimuli-responsive polymer cushions to tune the frictional drag between polymer cushion and mobile membranes with incorporated integrins to actively regulate lipid membrane characteristics. To attain this goal, a temperature- and pH-responsive polymer based on poly(N-isopropylacrylamide) copolymers containing varying amounts of carboxyl-group-terminated comonomers at different aliphatic spacer lengths (PNIPAAm-co-carboxyAAM) was surface-grafted to a poly(glycidyl methacrylate) anchorage layer. The swelling transitions were characterized using atomic force microscopy, ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and found to be tunable over a wide range of temperature and pH. In agreement with the behavior of the polymers in solution, longer alkyl spacers decreased the phase transition temperature T(P) and higher contents of carboxylic acid terminated comonomers increased T(P) at alkaline conditions and decreased T(P) at acidic conditions. Remarkably, the point where the degree of carboxyl group deprotonation balances the T(P)-lowering effect of the alkyl spacer was distinctive for each alkyl spacer length.
These findings illustrate how the local and global balance of hydrophilic and hydrophobic interactions along the copolymer chain allows to adjust the swelling transition to temperatures below, comparable, or above those observed for PNIPAAm homopolymers. Additionally, it could be shown that surface-grafting leads to a decrease in T(P) for PNIPAAm homopolymers (7°C) and copolymers (5°C - 10°C). The main reason is the increase in local polymer concentration of the swollen film constrained by dense surface anchorage in comparison to the behavior of dilute free chains in solution. In accordance with the Flory-Huggins theory, T(P) decreases with increasing concentration up to the critical concentration. Biological functionalization of the PNIPAAm-co-carboxyAAm thin films was demonstrated for the cell adhesion ligand peptide cRGD via carbodiimide chemistry to mimic extracellular binding sites for the cell adhesion receptors integrin.
The outcome of QCM-D measurements of cRGD-functionalized surfaces showed a maintained stimuli-responsiveness with slight reduction in T(P). A drying/rehydration procedure of a 9:1 lipid mixture of the cationic lipid dioleoyl-trimethylammoniumpropane (DOTAP) and the zwitterionic dioleoyl-phosphatidylcholine (DOPC) was utilized to form lipid bilayer membranes on PNIPAAm-co-carboxyAAM cushions. Fluorescence recovery after photobleaching (FRAP) revealed that lipid mobility was distinctively higher (6.3 - 9.6) µm2 s-1 in comparison to solid glass support ((3.0 - 5.9) µm2 s-1). In contradiction to the initial expectations, modulation of temperature and pH led to poor variations in lipid mobility that did not correlate with the PNIPAAm cushion swelling state. The results suggested a weak coupling of the lipid bilayer with PNIPAAm polymer cushions that can be slightly tuned by electrostatic interactions.
The transmembrane adhesion receptor alpha(5)beta(1) integrin was reconstituted into liposomes consisting of DOPC/sphingomyelin/cholesterol 2:2:1 for the formation of polymer cushioned bilayers. PNIPAAm- co-carboxyAAM and maleic acid (MA) copolymers were used as cushions, both with the option for cRGD functionalization. On the MA copolymer cushions, fusion of proteoliposomes resulted in supported bilayers with mobile lipids as confirmed by FRAP. However, incorporated integrins were immobile. In an attempt to explain this observation, the medium-sized cytoplasmic integrin domain was accounted to hamper the movement by steric interactions with the underlying polymer chains in conjunction with electrostatic interactions of the cationic cytoplasmic domain with the oppositely charged MA copolymer.
On the PNIPAAm-co-carboxyAAM cushion only a drying/rehydration procedure lead to bilayer formation. However, again the integrins were immobile, presumably due to the harsh treatment during preparation. Nevertheless, the results of the investigated set of PNIPAAm copolymer films suggest their application as temperature- and pH-responsive switchable layers to control interfacial phenomena in bio-systems at different physiological conditions. The PNIPAAm-co-carboxyAAm cushioned bilayer system represents a promising step towards extrinsically controlled membrane – substrate interactions.
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