Ein Modell der lichtgetriebenen Protonenpumpe Bakteriorhodopsin postulierte die direkte Beteiligung der Wasserstoffe in der 4-Stellung des Cyclohexenringes des Retinalchromophors an dem Vorgang der Protonenverschiebung. Mittels Blockaden der Retroform-Bildung von Retinal durch chemische Modifizierungen des Cyclohexenringes (4-Hydroxy-Retinal, 5,6-Epoxy-Retinal) konnten nach Einbau der modifizierten Moleküle in die isolierte Purpurmembran und nach Zugabe zu Halobakterien mit unterdrückter Retinalsynthese die direkte Beteiligung des Cyclohexenringes an der Protonenpumpe mit großer Wahrscheinlichkeit ausgeschlossen werden.

Universiẗat, Diss., 2001--München.

Techn. Universiẗat, Diss., 1999--Darmstadt.

Universiẗat, Diss., 2003--Dortmund.

Stuttgart, Univ., Diss., 2001.

Universiẗat, Diss., 2000--München.

Darmstadt, Techn. Univ., Diss., 2001. / Dateiformat: tar.gz, Dateien im PDF-Format

Universiẗat, Diss., 2001--Dortmund.

Die lichtgetriebene Protononenpumpe Bacteriorhodopsin (BR) aus Halobacterium salinarum stellt den Prototyp lichtgetriebener Protonenpumpen dar und wurde durch strukturelle und spektroskopische Untersuchungen als einfaches Modellsystem für Protonentransferschritte in Proteinen etabliert. Aufgrund der schlechten heterologen Expression von BR in Wirtszellen konnten elektrophysiologische Untersuchungen unter kontrollierter Membranspannung jedoch nur vereinzelt durchgeführt werden und erlaubten kaum Rückschlüsse zum Einfluss einzelner Aminosäuren auf die Pumpkraft des Proteins. Das dem BR nahverwandte Coccomyxa subellipsoidea Rhodopsin (CsR) hingegen zeigte gegenüber BR etwa 8-fach größere Photoströme in elektrophysiologischen Messungen in Xenopus laevis Oozyten und ermöglichte so eine umfangreiche elektrophysiologische Charakterisierung. Dabei konnten Unterschiede zu BR, wie etwa die erhöhte Spannungsabhängigkeit oder die erhöhte Toleranz der Pumpkraft gegenüber dem extrazellulären pH, auf Grundlage des Sequenzvergleiches nicht geklärt werden. Entsprechend wurden, um weitere mechanistische Details zu klären, im Rahmen dieser Arbeit spektroskopische und röntgenkristallographische Untersuchungen an CsR vorgenommen. Dabei konnte eine hochauflösende Kristallstruktur von CsR gelöst werden, die es erlaubt, mechanistische Unterschiede zu anderen lichtgetriebenen Protonenpumpen auf strukturelle Unterschiede zurück zu führen. So konnte die erhöhte Spannungsabhängigkeit mit der besonderen Komposition des zytoplasmatischen Halbkanals und die erhöhte Toleranz gegenüber dem äußeren pH mit der einzigartigen Konfiguration des Protonenfreisetzungskomplexes in CsR assoziiert werden. Letztlich konnte auf Grundlage der Struktur CsR, durch rationales Design, in einen licht-induzierten Protonenkanal transformiert werden, der bereits bei physiologischen Bedingungen quasisymmetrische bidirektionale Ströme zeigt. / The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum represents the prototype of light-driven proton pumps and was established by structural and spectroscopic investigations as a simple model system for proton transfer steps in proteins. However, due to the poor heterologous expression of BR in host cells, electrophysiological investigations under controlled membrane potential could only be carried out in some cases and hardly allowed conclusions to be drawn about the influence of individual amino acids on the pumping force of the protein. Coccomyxa subellipsoidea rhodopsin (CsR), which is closely related to BR, showed about 8 times larger photocurrents in electrophysiological measurements in Xenopus laevis oocytes than BR and thus enabled an extensive electrophysiological characterization. Observed differences to BR, such as the increased voltage dependence or the increased tolerance of the pumping force to the extracellular pH, could not be clarified solely on the basis of the sequence comparison. Accordingly, in order to clarify further mechanistic details, spectroscopic and X-ray crystallographic investigations on CsR were carried out within the scope of this work. A high-resolution crystal structure of CsR was solved, which allows to link mechanistic differences to other light-driven proton pumps to structural differences. Thus, the increased voltage dependence could be addressed by the special composition of the cytoplasmic half channel and the increased tolerance to the external pH could be associated with the unique configuration of the proton release complex in CsR. Finally, by a structure-based rational design approach, CsR was transformed into a light-induced, which shows almost symmetrical bidirectional currents already under physiological conditions.

Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage.