title: Single-Molecule Fluorescence Studies of Fast Protein Folding.
abstract: Watching the conformational wanderings of protein molecules during their search for their native structure is a holy grail for protein folding experimentalists. Such capability is essential to provide reality checks to the complex mechanistic heterogeneity that theory and molecular simulations predict. Single-molecule fluorescence resonance energy transfer (SM-FRET) is an attractive technique to meet that end, but its time resolution was insufficient for the microsecond motions of folding proteins. The temporal resolution of SM-FRET pivots on how quickly photons emitted by an individual molecule can be collected in sufficient numbers as to minimize statistical shot noise. Recent multipronged advances in that front have led us to gain first access to fast folding dynamics. Center stage is the implementation of photochemical strategies to quickly recover fluorophores from long-lasting dark states. Improvements in optics and novel procedures for data analysis in probabilistic terms are additional contributions. In parallel, noninvasive methods for fluorescent labeling and immobilization of proteins have also been implemented. Here we discuss all these exciting developments, providing experimental guidelines and procedural details for the implementation of fast SM-FRET experiments on proteins.
authors: Bioengineering Program, School of Engineering, University of California at Merced, Merced, CA, United States.
authors: Z
authors: Z
authors: Wang
authors: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors: L A
authors: LA
authors: Campos
authors: Bioengineering Program, School of Engineering, University of California at Merced, Merced, CA, United States; Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain. Electronic address: vmunoz3@ucmerced.edu.
authors: V
authors: V
authors: Muñoz
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins
keywords: Fast protein folding; Folding energy landscapes; Protein conformational motions; Single-molecule fluorescence spectroscopy; Single-molecule techniques