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.

Molecular dynamics of biomolecules, like proteins and nucleic acids dictate essential biological processes allowing life to function. They are involved in a vast number of cellular tasks including DNA replication, genetic recombination, transcription and translation, as well as signalling, translational motion, structure formation, biochemical synthesis, immune response, and many more. Developed over billions of years by evolution they constitute fine-tuned networks modulated by temperature and regulatory mechanisms. A better understanding of the thermodynamic fundamentals of inter- and intramolecular conformational changes can shed light on the underlying processes of diseases and enables the transfer of biological architectures, properties and compositions to nanotechnological applications. Dynamics of biomolecules occur on a wide range of timescales covering more than twelve orders of magnitude. Fluorescence spectroscopy techniques like time-correlated single photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), and immobilized and freely diffusing single-molecule Förster resonance energy transfer (FRET) spectroscopy represent powerful tools monitoring the dynamics at different ranges within this large span of timescales. However, a unified approach covering all biological relevant timescales remains a goal in the field of fluorescence spectroscopy. This would comprise a methodological workflow for qualitative and quantitative analysis of biomolecular dynamics ranging from nanoseconds to hours. In this work, a custom built single-molecule fluorescence spectroscopy set-up was constructed combining confocal single-molecule FRET spectroscopy with TCSPC, FCS and fluorescence anisotropy techniques for multiparameter fluorescence detection (MFD). The set-up allows the complementary observation of single-molecules over an extensive timescale ranging from fast reconfiguration dynamics of polymers (nanoseconds) to slow membrane protein folding (hours) without the need of molecular synchronization. Freely diffusing molecules enable high throughput measurements in heterogeneous membrane-mimetic and denaturing environments. Additionally, routines for data acquisition and processing were developed followed by the elaboration of a methodological workflow for the qualitative and quantitative analysis of biomolecular dynamics. Finally, the applicability was demonstrated on a big diversity of biological systems (DNA hairpin, Holliday junction, soluble and membrane proteins) in aqueous, membrane-mimetic and denaturing environments covering conformational dynamics from nanoseconds to hours.:Chapter 1: Introduction Chapter 2: Dynamics of Biomolecules 2.1 Dynamics of Nucleic Acids 2.1.1 DNA Hairpin Dynamics 2.1.2 Dynamics of Holliday Junctions 2.2 Dynamics of Proteins 2.2.1 Model Systems of Protein Folding Chapter 3: Fundamentals of Fluorescence Spectroscopy 3.1 Basics of Fluorescence 3.2 Förster Resonance Energy Transfer (FRET) Chapter 4: Multiparameter Fluorescence Detection 4.1 Single-Molecule FRET Spectroscopy 4.1.1 Confocal Microscopy 4.1.2 Freely Diffusing Molecules 4.1.3 Fluorescence Spectroscopy 4.2 Time-Correlated Single-Photon Counting (TCSPC) 4.3 Pulsed Interleaved Excitation (PIE) 4.4 Fluorescence Anisotropy 4.5 Fluorescence Correlation Spectroscopy (FCS) 4.6 MFD Setup 4.7 Analysis Software Chapter 5: Analysis of Molecular Dynamics 5.1 Sub-Microseconds – Peptide Chain Dynamics 5.1.1 Identification of Peptide Chain Dynamics 5.1.2 Quantification of Peptide Chain Dynamics 5.1.3 Discussion 5.2 Microseconds – Dynamics of Barrier Crossing 5.2.1 Maximum Likelihood Estimation of the Transition-Path Time 5.2.2 Quantification of the Upper Bound of the Transition-Path Time 5.2.3 Discussion 5.3 Milliseconds – Fast Protein Folding Dynamics 5.3.1 Correlation of the Relative Donor Lifetime (τD(A) / τD(0)) with FRET Efficiency (E) 5.3.2 Burst-Variance Analysis (BVA) 5.3.3 FRET-Two-Channel Kernel-Based Density Distribution Estimator (FRET-2CDE) 5.3.4 Estimation of the Conformational Relaxation Rate using Bin-Time Analysis 5.3.5 Extracting Folding Kinetics using the Three-Gaussian (3G) Approximation 5.3.6 Dynamic Probability Distribution Analysis (dPDA) 5.3.7 Folding and Unfolding Rate Estimation using a Maximum-Likelihood Estimator 5.3.8 Discussion 5.4 Milliseconds to Seconds – Stacking Dynamics of DNA 5.4.1 Identification of Dynamics on the Recurrence Timescale 5.4.2 Quantification of Dynamics on the Recurrence Timescale 5.4.3 Discussion 5.5 Minutes to Hours – Slow Protein Folding Dynamics 5.5.1 Identification of Slow Protein Folding Dynamics 5.5.2 Quantification of Slow Protein Folding Dynamics 5.5.3 Discussion Chapter 6: Conclusion and Outlook Chapter 7: Appendices 7.1 Derivation of Equation 4.6 (inspired by Daniel Nettels) 7.2 Protein sequences 7.3 Identification of dynamics on the recurrence timescale 7.4 Dependency of psame on the sample concentration 7.5 Effect of fluorescence quenching on MFD parameters Chapter 8: References / Biomoleküle, wie Proteine und Nukleinsäuren, sind essentielle Bausteine des Lebens und permanent an biologischen Prozessen beteiligt. Innerhalb der Zelle nehmen sie eine Vielzahl von Aufgaben wahr, darunter DNA-Replikation, genetische Rekombination, Transkription und Translation, sowie Signalübertragung, Transport, Strukturbildung, biochemische Synthese und Immunreaktion. In Milliarden von Jahren evolutionärer Entwicklung wurden biomolekulare Prozesse immer feiner aufeinander Abgestimmt. Um den zugrundeliegenden Mechanismus von Krankheiten besser zu Verstehen und um die einzigartigen Eigenschaften und Kompositionen biologischer Systeme auf nanotechnologische Anwendungen übertragen zu können, ist es unbedingt notwendig ein besseres Verständnis thermodynamischer Grundlagen inter- und intramolekularer Konformationsänderungen zu erlangen. Dabei finden sich Dynamiken von Biomolekülen über eine Zeitskale von mehr als zwölf Größenordnungen verteilt. Fluoreszenzspektroskopietechniken, wie zeitkorrelierte Einzel-photonenzählung (TCSPC), Fluoreszenzkorrelationsspektroskopie (FCS), und Förster-Resonanzenergietransfer (FRET)–Spektroskopie von immobilisierten und frei diffundierenden Molekülen, stellen leistungsfähige Werkzeuge dar, welche es ermöglichen Dynamiken in der den Techniken entsprechenden Zeitskala aufzulösen. Dennoch, besteht der dringende Bedarf nach einer einheitlichen Methode, der in der Fluoreszenzspektroskopie alle biologisch relevanten Zeitskalen abdeckt. Dies würde einen methodischen Workflow für die qualitative und quantitative Analyse der biomolekularen Dynamik von Nanosekunden bis Stunden bedeuten. In dieser Arbeit wurde ein speziell angefertigter Multiparamter-Fluoreszenzspektroskopie-Aufbau konstruiert, welcher die konfokale Einzelmolekül-FRET-Spektroskopie mit den TCSPC-, FCS- und Fluoreszenz-Anisotropie-Techniken kombiniert. Der Aufbau ermöglicht die Beobachtung komplementärer Eigenschaften von Einzelmolekülen über eine umfangreiche Zeitskala hinweg. Dynamiken von schnell rekonfigurierenden Polymeren (Nanosekunden) bis hin zu langsam faltenden Membranproteinen (Stunden) sind ohne molekulare Synchronisation möglich. Darüber hinaus, ermöglicht der Einsatz frei diffundierender Moleküle einen hohen Messdurchsatz und die Anwendung heterogener membranmimetischer und denaturierender Lösungen. Zusätzlich wurden Routinen zur Datenerfassung und -verarbeitung entwickelt, gefolgt von der Ausarbeitung eines methodischen Workflows zur qualitativen und quantitativen Analyse von biomolekularen Dynamiken. Abschließend wurde die Anwendbarkeit an fünf biologischen Modelsystemen (DNA-Haarnadel, Holliday-Junction, lösliche und Membranproteine) in wässrigen, membranmimetischen und denaturierenden Umgebungen demonstriert und alle biologisch relevanten Zeitskalen von Nanosekunden bis Stunden abgedeckt.:Chapter 1: Introduction Chapter 2: Dynamics of Biomolecules 2.1 Dynamics of Nucleic Acids 2.1.1 DNA Hairpin Dynamics 2.1.2 Dynamics of Holliday Junctions 2.2 Dynamics of Proteins 2.2.1 Model Systems of Protein Folding Chapter 3: Fundamentals of Fluorescence Spectroscopy 3.1 Basics of Fluorescence 3.2 Förster Resonance Energy Transfer (FRET) Chapter 4: Multiparameter Fluorescence Detection 4.1 Single-Molecule FRET Spectroscopy 4.1.1 Confocal Microscopy 4.1.2 Freely Diffusing Molecules 4.1.3 Fluorescence Spectroscopy 4.2 Time-Correlated Single-Photon Counting (TCSPC) 4.3 Pulsed Interleaved Excitation (PIE) 4.4 Fluorescence Anisotropy 4.5 Fluorescence Correlation Spectroscopy (FCS) 4.6 MFD Setup 4.7 Analysis Software Chapter 5: Analysis of Molecular Dynamics 5.1 Sub-Microseconds – Peptide Chain Dynamics 5.1.1 Identification of Peptide Chain Dynamics 5.1.2 Quantification of Peptide Chain Dynamics 5.1.3 Discussion 5.2 Microseconds – Dynamics of Barrier Crossing 5.2.1 Maximum Likelihood Estimation of the Transition-Path Time 5.2.2 Quantification of the Upper Bound of the Transition-Path Time 5.2.3 Discussion 5.3 Milliseconds – Fast Protein Folding Dynamics 5.3.1 Correlation of the Relative Donor Lifetime (τD(A) / τD(0)) with FRET Efficiency (E) 5.3.2 Burst-Variance Analysis (BVA) 5.3.3 FRET-Two-Channel Kernel-Based Density Distribution Estimator (FRET-2CDE) 5.3.4 Estimation of the Conformational Relaxation Rate using Bin-Time Analysis 5.3.5 Extracting Folding Kinetics using the Three-Gaussian (3G) Approximation 5.3.6 Dynamic Probability Distribution Analysis (dPDA) 5.3.7 Folding and Unfolding Rate Estimation using a Maximum-Likelihood Estimator 5.3.8 Discussion 5.4 Milliseconds to Seconds – Stacking Dynamics of DNA 5.4.1 Identification of Dynamics on the Recurrence Timescale 5.4.2 Quantification of Dynamics on the Recurrence Timescale 5.4.3 Discussion 5.5 Minutes to Hours – Slow Protein Folding Dynamics 5.5.1 Identification of Slow Protein Folding Dynamics 5.5.2 Quantification of Slow Protein Folding Dynamics 5.5.3 Discussion Chapter 6: Conclusion and Outlook Chapter 7: Appendices 7.1 Derivation of Equation 4.6 (inspired by Daniel Nettels) 7.2 Protein sequences 7.3 Identification of dynamics on the recurrence timescale 7.4 Dependency of psame on the sample concentration 7.5 Effect of fluorescence quenching on MFD parameters Chapter 8: References

Funktionalisierte organische Perylenbisimidfarbstoffe (PBI) und aus Cadmiumselenid bestehende Halbleiternanokristalle werden hinsichtlich physikalischer sowie chemischer Wechselwirkungsprozesse miteinander und mit ihrer Umgebung mittels zeitaufgelöster optischer Spektroskopie untersucht. Im Mittelpunkt der Studien an diesem organisch/anorganischen Modellsystem nanoskopischer Größe steht die Aggregatbildungskinetik und die Identifikation und Quantifizierung von Transferpozessen. Die Anbindung der gut löslichen PBI-Farbstoffe an die Oberfläche solcher Halbleiternanokristalle mittels spezieller Ankergruppen wird durch Selbstorganisation in Lösung realisiert. Die Kombination von Absorptions- und zeitaufgelöster Fluoreszenzspektroskopie zeigt einen unterschiedlich starken Einfluss von Liganden und Farbstoffen auf die Fluoreszenzlöschung der Nanokristalle und belegt, dass Resonanzenergietransfer zum Farbstoff nur in sehr geringem Maße die physikalische Ursache der Fluoreszenzlöschung ist. Die Anzahl adsorbierter Farbstoffe und die Stärke der Fluoreszenzlöschung eines einzelnen Farbstoffmoleküls werden aus zeitaufgelösten Einzelmolekülexperimenten an immobilisierten Emittern gewonnen, welche den direkten spektroskopischen Zugang zur Verteilung gebundener und freier Farbstoffe/Nanokristalle erlaubt. Darüber hinaus werden ankergruppen- und umgebungsspezifische Einflüsse auf die Konformations- und Orientierungsdynamik von Perylenbisimidmolekülen dargestellt. Abschließend werden photo-physikalische Gemeinsamkeiten chemisch unterschiedlich hervorgerufener Fluoreszenzlöschungsprozesse herausgearbeitet und im Kontext von Einzelkristall-Blinkprozessen diskutiert.