Probing Conformational Dynamics of Biomolecules using Single-Photon and Single-Molecule Spectroscopy

Abstract

Biomolecules are dynamic entities that can switch between different structural states. These structural changes are often tied to their biological function within the context of a cells metabolism. Resolving and understanding changes in a biomolecules structure is therefore essential for gaining insights into its function and regulation. Standard methods of structural biology provide a detailed, but often static, picture of a biomolecule. Critical information on how biomolecules change shape over time is difficult to extract or cannot be extracted from this type of data, leaving a critical gap of knowledge on the structure-function relationship of biomolecules. In this thesis, single-molecule Förster resonance energy transfer spectroscopy (smFRET) is used to directly resolve structural dynamics of biomolecules in real time. Contrary to methods that measure a bulk of molecules, single-molecule-resolved methods capture the instantaneous state of individual molecules. This single-molecule view avoids averaging and makes the full range of states directly observable. Changes in a biomolecules structure can be detected by FRET, which is a physical phenomenon that allows measuring distance changes within a few nanometers, compatible with distance changes that occur within biomolecules.