Studying protein-folding dynamics using single-molecule fluorescence methods

Abstract

Protein folding is an ancient biological problem, and to address this problem, high throughput, sophisticated, and sensitive detection technique is required. Single-molecule spectroscopy is such a state-of-the-art approach to study the heterogeneous system of protein folding. With the advancement of confocal setup, single-molecule (SM) spectroscopy has been extremely useful in studying transient, thermodynamically unstable sub-populations of the folding process for both globular and intrinsically disordered proteins. Fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET) are the two main single-molecule techniques frequently used in protein conformational study. In this chapter, we highlight the basic theoretical principle of single-molecule technique emphasizing FCS, FRET, and SM microscopy adorned with some experimental examples.