Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome.

Protein folding is a fundamental biological process of great significance for cell function and life-related processes. Surprisingly, very little is presently known about how proteins fold in vivo. The influence of the cellular environment is of paramount importance, as molecular chaperones, the ribosome, and the crowded medium affect both folding pathways and potentially even equilibrium structures. Studying protein folding in physiologically relevant environments, however, poses a number of technical challenges due to slow tumbling rates, low concentrations and potentially non-homogenous populations. Early work in this area relied on biological assays based on antibody recognition, proteolysis, and activity studies. More recently, it has been possible to directly observe the structure and dynamics of nascent polypeptides at high resolution by spectroscopic and microscopic techniques. The fluorescence depolarization decay of nascent polypeptides labeled with a small extrinsic fluorophore is a particularly powerful tool to gain insights into the dynamics of newly synthesized proteins. The fluorophore label senses both its own local mobility and the motions of the macromolecule to which it is attached. Fluorescence anisotropy decays can be measured both in the time and frequency domains. The latter mode of data collection is extremely convenient to capture the nanosecond motions in ribosome-bound nascent proteins, indicative of the development of independent structure and folding on the ribosome. In this review, we discuss the theory of fluorescence depolarization and its exciting applications to the study of the dynamics of nascent proteins in the cellular environment.