Click chemistry is a molecular synthesis strategy based on reliable, highly selective reactions with thermodynamic driving forces typically in excess of 20 kcal mol-1. The 1,3-dipolar cycloaddition of azides and alkynes developed by Rolf Huisgen saw dramatic rate acceleration using Cu(I) as a catalyst in 2002 reports by Barry Sharpless and Morten Meldal enabling its click chemistry eligibility. Since these seminal reports, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has become the quintessential click reaction finding diverse utility. The popularity of the CuAAC has naturally led to interest in new catalyst systems with improved efficiency, robustness, and reusability with particular focus on nanomaterial catalysts, a common trend across the field of catalysis. The high surface area of nanomaterials lends to their efficacy as colloidal and heterogeneous nanocatalysts, but the latter boasts the added benefit of easy separation and recyclability. With any heterogeneous catalyst, a common question arises as to whether the active catalyst species is truly heterogeneous or rather homogeneous through metal ion leaching. Differentiating these processes is critical, as the latter would result in reduced efficiency, higher cost, and inevitable environmental and heath side effects. This thesis explores the CuAAC from an interdisciplary approach. First as a synthetic tool, applying CuAAC-formed triazoles as functional, modular building blocks in the synthesis of optical cation sensors by combining azide and alkyne modified components to create a series of sensors selective for different metal cations. Next, single-molecule spectroscopy techniques are employed to observe the CuNP-catalyzed CuAAC in real time. Combining bench-top techniques with single-molecule microscopy to monitor single-catalytically generated products proves to be an effective method to establish catalysis occurs directly at the surface of copper nanoparticles, ruling out catalysis by ions leached into solution. This methodology is extended to mapping the catalytic activity of a commercial heterogeneous catalyst by applying super-localization analysis of single-catalytic events. The approach detailed herein is a general one that can be applied to any catalytic system through the development of appropriate probes. This thesis demonstrates single-molecule microscopy as an accessible, effective, and unparalleled tool for exploring the catalytic activity of nanomaterials by monitoring single-catalytic events as they occur.

Within the last years, e.g. by investigating the fluorescence of single molecules in biological cells, remarkable progress has been made in cell biology extending conventional ensemble techniques concerning temporal / spatial resolution and the detection of particle subpopulations [82]. In addition to employing single fluorophores as "molecular beacons" to determine the position of biomolecules, single molecule fluorescence studies allow to access the photophysical dynamics of genetically encoded fluorescent proteins itself. However, in order to gain statistically consistent results, e.g. on the mobility behavior or the photophysical properties, the fluorescence image sequences have to be analyzed in a preferentially automated and calibrated (non-biased) way. In this thesis, a single molecule fluorescence optical setup was developed and calibrated and experimental biological in-vitro systems were adapted to the needs of single molecule imaging. Based on the fluorescence image sequences obtained, an automated analysis algorithm was developed, characterized and its limits for reliable quantitative data analysis were determined. For lipid marker molecules diffusing in an artifcial lipid membrane, the optimum way of the single molecule trajectory analysis of the image sequences was explored. Furthermore, effects of all relevant artifacts (specifically low signal-to-noise ratio, finite acquisition time and high spot density, in combination with photobleaching) on the recovered diffusion coefficients were carefully studied. The performance of the method was demonstrated in two series of experiments. In one series, the diffusion of a fluorescent lipid probe in artificial lipid bilayer membranes of giant unilamellar vesicles was investigated. In another series of experiments, the photoconversion and photobleaching behavior of the fluorescent protein Kaede-GFP was characterized and protein subpopulations were identified.

Confocal single-molecule fluorescence is a powerful tool for monitoring conformational dynamics, and has provided new insight into the enzymatic activities of complex biological molecules such as DNA and RNA polymerases. Though useful, such studies are typically qualitative in nature, and performed almost exclusively in highly purified, in vitro settings. In this work, I focus on improving the methodology of confocal single-molecule fluorescence in two broad ways: (i) by enabling the quantitative identification of molecular dynamics in proteins and nucleic acids in vitro, and (ii) developing the tools needed to perform these analyses in vivo. Toward the first goal, and together with several colleagues, I have developed three novel methods for the quantitative identification of dynamics in biomolecules: (i) Burst Variance Analysis (BVA), which unambiguously identifies dynamics in single-molecule FRET experiments; (ii) Dynamic Probability Density Analysis (PDA), which hypothesis-tests specific kinetic models against smFRET data and extracts rate information; and (iii) a novel molecular counting method useful for studying single-molecule thermodynamics. We validated these methods against Monte Carlo simulations and experimental DNA controls, and demonstrated their practical application in vitro by analyzing the “fingers-closing” conformational change in E.coli DNA Polymerase I; these studies identified unexpected conformational flexibility which may be important to the fidelity of DNA synthesis. To enable similar studies in the context of a living cell, we generated a nuclease-resistant DNA analogue of the Green Fluorescent Protein, or “Green Fluorescent DNA,” and developed an electroporation method to efficiently transfer it into the cytoplasm of E.coli. We demonstrate in vivo confocal detection of smFRET from this construct, which is both bright and photostable in the cellular milieu. In combination with PDA, BVA and our novel molecular counting method, this Green Fluorescent DNA should enable the characterization of DNA and protein-DNA dynamics in living cells, at the single-molecule level. I conclude by discussing the ways in which these methods may be useful in investigating the dynamics of processes such as transcription, translation and recombination, both in vitro and in vivo.

Droplet Interface Bilayers (DIBs) have a number of attributes that distinguish them from conventional artificial lipid bilayers. In particular, the ability to manipulate bilayers mechanically is explored in this thesis. Directed bilayer area changes are used to make precise measurements of the specific capacitance of DIBs and to control the two dimensional concentration of a membrane protein reconstituted in the bilayer. Chapter 1 provides a general introduction to the role of the lipid membrane en- vironment in the function of biological membranes and their integral proteins. An overview of model lipid bilayer systems is given. Chapter 2 introduces work carried out in this laboratory previously and illustrates the experimental setup of DIBs. Some important bilayer biophysical concepts are covered to provide the theoretical background to experiments in this and in later chapters. Results from the characterisation of DIBs are reported, and an account of the development of methods to manipulate the bilayer by mechanical means is given. Chapter 3 describes experiments that apply bilayer area manipulation in DIBs to achieve precise measurement of specific capacitance in a range of lipid systems. Chapter 4 reports results from experiments investigating the response of bilayer specific capacitance to an applied potential. Chapter 5 covers the background and experimental setup for total internal fluo- rescence microscopy experiments in DIBs and describes the expression, purification and characterisation of the bacterial β-barrel membrane protein pore α-Hemolysin. Chapter 6 describes experiments that apply the mechanical manipulation of bilayer area in DIBs to the corralling and control of the surface density of α-Hemolysin.

Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.