Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules (original) (raw)
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Soft Matter, 2011
Due to their high sensitivity and specificity fluorescence based single molecule techniques offer the possibility to study individual molecules (e.g., proteins or protein complexes) in situ in their cellular context. Recent progress in instrumentation and in sample preparation provides an increasingly better accessibility to more complex molecular assemblies. These assemblies mimic the natural cellular environmental conditions and at the same time allow sophisticated studies on proteins of interest. This review gives a brief introduction to single molecule fluorescence techniques and presents some selected applications on protein folding and on complex formation of membrane proteins.
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Biochemical Society Transactions, 2004
Single-molecule fluorescence has the capability to detect properties buried in ensemble measurements and, hence, provides new insights about biological processes. Ratiometric methods are normally used to reduce the effects of excitation beam inhomogeneity. Fluorescence resonance energy transfer is widely used but there are problems in inserting the fluorophores in the correct position on the biomolecule, particularly if the structure is not known. We have recently developed two-colour coincidence single-molecule fluorescence that addresses this problem. This method can be used to determine quantitatively the multimerization states of biomolecules, in solution without separation. The future prospects of single-molecule fluorescence as applied to biological molecules are discussed.
Fluorescence spectroscopy of single biomolecules
1999
Abstract Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging.
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Annual Review of Biochemistry, 2008
Ever since their introduction two decades ago, single-molecule (SM) fluorescence methods have matured and branched out to address numerous biological questions, which were inaccessible via ensemble measurements. Among the current arsenal, SM fluorescence techniques have capabilities of probing the dynamic interactions of nucleic acids and proteins via Förster (fluorescence) resonance energy transfer (FRET), tracking single particles over microns of distances, and deciphering the rotational motion of multisubunit systems. In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology.
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One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of this chapter. Fluorescence methods provide the sensitivity required to study biology at the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, first a general overview is given of single-molecule fluorescence microscopy. I start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, the fluorescent probes and the equipment required to visualize them on the single-molecule level will be reviewed in more detail. The chapter ends with a description of parameters measurable with such approaches, ranging from protein counting and tracking, to distance measurements with Förster Resonance Energy Transfer (see also Chapter 2.3) and orientation measurements with fluorescence polarization.
One-pot labeling and purification of peptides and proteins with fluorescein maleimide
Tetrahedron Letters, 2003
Fluorescein labeling of peptides and proteins is required for numerous biophysical or biological experiments such as fluorescence microscopy, fluorescence resonance energy transfer (FRET) or fluorescence imaging. The commonly used strategy relied on the coupling of the dye reagent followed by a gel filtration to recover the labeled molecule. Here we report a simplified method for the labeling of peptides and proteins on a cysteine residue and their purification. The method is based on the precipitation of peptides and proteins in acetone, fluorescein maleimide being soluble in this solvent. The excess of dye is fully eliminated after a couple of acetone washes and the precipitated peptide or protein is readily recovered.
Choosing the Probe for Single-Molecule Fluorescence Microscopy
International Journal of Molecular Sciences
Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.g., to fluorophore aspecific interactions, brightness, photostability, blinking, and emission and excitation spectra. In particular, these spectra and the excitation source are interdependent, and the latter affects the autofluorescence of sample substrate, medium, and/or biological specimen. Here, we review these and other critical points for fluorophore selection in single-molecule microscopy. We also describe the possible kinds of fluorophores and the microscopy techniques based on single-molecule fluorescence. We explain the importance and impact of the vari...
Fluorescence correlation spectroscopy for the detection and study of single molecules in biology
BioEssays, 2002
The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution-based single molecule technique, fluorescence correlation spectroscopy (FCS). This time-averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical confidence. FCS has proven to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual-color cross-correlation and two-photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research.
Scientific Reports, 2017
In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins. In recent years, there has been a rapid increase in both the development and utilization of fluorescence probes as molecular reporters for protein dynamics in cellular biology 1. Fusion-tag proteins, such as green fluorescent proteins (GFP), acyl carrier proteins (ACP), SNAP-, Halo-and CLIP-tags, have grown in popularity because they can be inserted at the genetic level of the protein of interest (POI) at either the Nor C-terminus 2-7. Fluorescent proteins (FPs) have their chromophores buried within a beta-barrel folded structure, whereas the fusion methods place synthetic fluorophores onto active sites near the surface of the fusion-tag when attached to the POI, and are often facilitated by biochemical catalytic conditions 2,3,8,9. A variety of FRET sensors were developed using FP colorimetric variants and fusion-tags to probe complex cellular processes 1,10,11. These studies include post-translational modifications, conformational changes, metal-ion binding and protein-protein interactions 2. FP variants however, are less bright and photostable than synthetic fluorophores (e.g., AlexaFluor ® dyes) and are known to show large variations in brightness due to differential chromophore maturation 2,12. Fusion-tags can alleviate this concern through the incorporation of high-quality fluorophores, however, drawbacks pertaining to bulkiness and steric hindrance remain. These factors limit both the kinetic and spatial resolution 9. Fusion-tag proteins are typically comprised of 77-300 amino acids (~8 to 30 kDa), which, depending on the insertion point, could result in loss of functionality of the tagged protein 7,8. Consequently, genetic-insertion of these probes have been limited to either the Nor the C-terminus, restricting the dynamical and structural information that can be accessed. Short peptide motifs consisting of less than 12 amino acids have recently been