Single-Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

1.1. Aims of This Review Single-molecule measurements provide unique information on heterogeneous populations of molecules: They give access to the complete distribution of observables (rather than only their first moments), allow discrimination between static and dynamic heterogeneity of their properties, and enable the detection of rare events or a succession of events hidden by ensemble averaging and the impossibility to synchronize molecules.1–8 Single-molecule methods have now pervaded several disciplines, in particular chemistry, evolving from a stage of proof-of-principle experiments to decisive research and discovery tools. A literature database search with the keyword “single molecule” gave over 5000 references at the time of this writing. This renders the prospect of an exhaustive discussion of current single-molecule work rather daunting. It proves, however, that single-molecule methods have gained the status of established techniques in various scientific fields and continue to propagate to new ones. Therefore, it seemed appropriate for a review of single-molecule methods in chemistry to provide a rapid description of the main technical approaches and focus on a few illustrative examples of their elucidative power. Even such an endeavor would have resulted in a heteroclite description of research on topics as diverse as quantum electrodynamics, low temperature and room temperature experiments on nanoparticles and organic or biological molecules, micromechanical manipulation, or fluorescence spectroscopy, among many others. Such an accumulation would have been of little interest, once the basic principles underlying each technique had been explained. It seemed, therefore, more appropriate to describe applications of a unique set of methods (fluorescence spectroscopy) to biochemical questions and, more specifically, the elucidation of protein structure, dynamics, and function. Protein structure and function are intimately related, and a large amount of single-molecule studies have been performed to elucidate the nature and role of conformational changes in protein or enzyme functions. These questions are best studied when methods have been validated on model systems, and we will delve into some simple examples of such systems to illustrate concepts, which are used in more sophisticated and ambitious studies. Another important aspect of protein science is the mechanism of protein structure formation (and loss thereof), i.e., protein folding and unfolding. Single-molecule methods have begun to yield very interesting results in this domain, and undoubtedly, more will follow. We have thus made this promising field one of the focuses of our review. The review is organized as follows. We will first define the questions that have been studied so far at the ensemble level and that are now being addressed with single-molecule fluorescence spectroscopy. The next section presents a summary of basic concepts of fluorescence spectroscopy and briefly reviews recent developments in single-molecule analysis, to serve as a glossary for all experimental approaches described throughout the remainder of this review. We then turn to applications of single-molecule fluorescence resonance energy transfer (FRET) to study polypeptide chain collapse in small single-domain proteins under equilibrium conditions. We provide some examples on how to extract dynamic information from single molecules, namely, distance distributions within conformational subpopulations of proteins in the framework of protein folding and in enzymes. These aspects are divided into two parts: studies based on FRET and studies relying on fluorescence quenching. The last part of this review addresses recent studies of protein folding dynamics under nonequilibrium conditions. We conclude with general remarks and an overview of future prospects of these methods.