Abstract
Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid‐liquid phase separation. The so‐called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein “structure‐function paradigm,” as their functions do not necessarily involve well‐defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single‐molecule fluorescence methods can overcome this ensemble‐average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single‐molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in‐depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel
Highlights
The purpose of this article is to outline a general protocol for implementing singlemolecule fluorescence (Förster resonance energy transfer, or FRET) techniques to study the conformational populations, binding interactions, and dynamics of proteins that do not possess a well-defined tertiary structure—so-called intrinsically disordered proteins (IDPs)
Single-molecule fluorescence methods are suited to IDP research, as they allow only a handful of proteins to be detected at the same time
Further purify the labeled proteins by HPLC or high-resolution ionexchange chromatography. This is especially useful if the labeling efficiency is low, which would be indicated by the measurement of a low yield in step 8 above for the donor-labeled protein, or by a large relative population of the “zero-peak” in the single-molecule Förster resonance energy transfer (smFRET) histogram for the donor-acceptor-labeled protein
Summary
The purpose of this article is to outline a general protocol for implementing singlemolecule fluorescence (Förster resonance energy transfer, or FRET) techniques to study the conformational populations, binding interactions, and dynamics of proteins that do not possess a well-defined tertiary structure—so-called intrinsically disordered proteins (IDPs). Site-specific unnatural amino acid (uAA) incorporation in conjunction with click chemistry labeling has become a popular approach for labeling proteins of interest for single-molecule fluorescence studies. This approach takes advantage of orthogonal tRNA and aminoacyl tRNA synthetase pairs that can recognize a specific uAA and incorporate it into the protein sequence in response to the presence of a unique codon. Many commercially available small-molecule dyes with different reactive handles fulfill the primary requirements for suitability for single-molecule measurements: high quantum yield and photostability These fluorophores span a wide range of excitation wavelengths, which is a crucial factor to consider in designing twoand multicolor experiments. Because the setup requires numerous components to build, a protocol for instrument building is outside the scope of this
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