Click Strategies for Single-Molecule Protein Fluorescence

Abstract

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

“…Structural changes in different regions of a protein also may be combined with dynamic interfacial, as well as functional, measurements, allowing direct conclusions to be drawn with respect to ways in which protein conformation, dynamics, and activity are connected. Although site-specific labeling methods have been used with FRET to monitor conformational changes in single protein molecules in bulk solution (17)(18)(19) or covalently tethered to surfaces (20)(21)(22), such methods have not been applied to the analysis of proteins undergoing dynamic adsorption, desorption, and diffusion while interacting directly with material interfaces. SM-FRET tracking, with temporal resolution of 100 ms, has previously been used to monitor dynamic changes in conformation of mobile DNA at the solution-surface interface (23,24), using high-throughput tracking algorithms that permit the observation of 10 4 -10 6 molecules.…”

Single-molecule FRET and crosslinking studies in structural biology enabled by noncanonical amino acids

“…One major complication is that investigators typically report labeling yields for proteins from a variety of expression hosts under very different conditions. To address this issue in part, in a systematic study by Milles et al [22 ] several ncAAs were expressed in a diverse set of proteins, and their corresponding labeling reactions were analyzed with respect to their performance in high-resolution smFRET experiments. Furthermore, the labeling chemistry can affect the local environment and properties of the FRET measurement to an extent that should not be ignored if high-resolution measurements are desired.…”

“…Using the first strategy, the high expression yields achievable using PrK incorporation allowed the design of a onestep FRET-pair-labeling strategy for large and cysteinecontaining proteins, such as RanBP3 (i.e., it is not a just a model system) [22 ]. This strategy gave the authors full freedom to place the labeling sites throughout the protein by incorporating two PrK residues in response to two Amber codons included at the desired sites in the gene sequence for RanBP3.…”

Fluorescently labeled recombinant RNAP system to probe archaeal transcription initiation

“…Over the last years, the UAA toolkit has been continually extended and improved [39]. Most frequently, UAAs suitable for subsequent click reaction or the StaudingerBertozzi ligation are incorporated into the protein of interest [40,41]. Introduction of any UAA during recombinant protein expression usually relies on the suppression of a stop codon (for recent reviews see [42][43][44]).…”