Protein Labeling Strategies Research Articles

Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells.

We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells. These custom-designed, photo-caged Q-rhodamines and fluoresceins are cell-permeable, bright and localize specifically to intracellular targets. We utilized established orthogonal protein labeling strategies to precisely attach the photoactivatable fluorophores to proteins with subsequent activation of fluorescence by irradiation with UV light. That way, diffusive cytosolic proteins, histone proteins as well as filigree mitochondrial networks and focal adhesion proteins were visualized inside living cells. We applied the new photoactivatable probes in inverse fluorescence recovery after photo-bleaching (iFRAP) experiments, gaining real-time access to protein dynamics from live biological settings with resolution in space and time. Finally, we used the caged Q-rhodamine for photo-activated localization microscopy (PALM) on both fixed and live mammalian cells, where the superior molecular brightness and photo-stability directly resulted in improved localization precisions for different protein targets.

ChemInform Abstract: Selective Chemical Labeling of Proteins

AbstractReview: 216 refs.

Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET.

Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and closed conformations of DNA polymerases, our smFRET assays utilizing doubly labeled variants of Escherichia coli DNA polymerase I were pivotal in identifying and characterizing a partially closed conformation as a primary checkpoint for nucleotide selection. Here, we provide a comprehensive overview of the methods we used for the conformational analysis of wild-type DNA polymerase and some of its low-fidelity derivatives; these methods include strategies for protein labeling and our procedures for solution-based single-molecule fluorescence data acquisition and data analysis. We also discuss alternative single-molecule fluorescence strategies for analyzing the conformations of DNA polymerases in vitro and in vivo.

Selective chemical labeling of proteins.

Over the years, there have been remarkable efforts in the development of selective protein labeling strategies. In this review, we deliver a comprehensive overview of the currently available bioorthogonal and chemoselective reactions. The ability to introduce bioorthogonal handles to proteins is essential to carry out bioorthogonal reactions for protein labeling in living systems. We therefore summarize the techniques that allow for site-specific "installation" of bioorthogonal handles into proteins. We also highlight the biological applications that have been achieved by selective chemical labeling of proteins.

A Covalent Approach for Site‐Specific RNA Labeling in Mammalian Cells

AbstractAdvances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site‐ and sequence‐specific covalent labeling of RNAs in mammalian cells by using tRNAIle2‐agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide‐bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site‐specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.

A covalent approach for site-specific RNA labeling in Mammalian cells.

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site- and sequence-specific covalent labeling of RNAs in mammalian cells by using tRNA(Ile2) -agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide-bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site-specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.

Illuminating biological processes through site-specific protein labeling.

Coupling genetically encoded peptide tags or unnatural amino acids (UAAs) with bioorthogonal reactions allows for precise control over the protein-labeling sites as well as the wide choice of labeling dyes. However, the value of these site-specific protein labeling strategies in a real biology setting, particularly their advantages over conventional labeling methods including fluorescent proteins (FPs), remains to be fully demonstrated. In this tutorial review, we first introduce various strategies for site-specific protein labeling that utilize artificial peptide sequences or genetically encoded UAAs as the labeling handle. Emphasis will be placed on introducing the advantages of protein site-specific labeling techniques as well as their applications in solving biological problems, particularly as to why a site-specific protein labeling approach is needed. Finally, beyond the widely used single site-specific labeling methods, the recently emerged dual site-specific protein labeling strategies will be introduced together with their fast-growing potential in illustrating biological processes.

Coumarin-based fluorogenic probes for no-wash protein labeling.

A fluorescent protein-labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.

Excited state photoreaction between the indole side chain of tryptophan and halocompounds generates new fluorophores and unique modifications.

Photoreaction of indole containing compounds with chloroform and other trichlorocompounds generates products with redshifted fluorescence. In proteins, this reaction can be used for the fluorescent detection of proteins. Little characterization of products generated through the photochemical reaction of indoles with halocompounds has been done, yet is fundamental for the development of other fluorophores, protein labeling agents, and bioactive indole derivatives. Here, we have characterized which isomers form in the photoreaction between tryptophan and chloroform using (1)H-NMR of tryptophan and methylated derivatives to reveal that the two major products that are formed result from modification at the 4- and 6-carbon positions of the indole ring. Reaction at position 6 generates 6-formyl tryptophan and the reaction at position 4 generates an imine because the formyl derivative that is initially formed reacts further with the tryptophan amine group. The spectroscopic properties and product molecular weights of photoproducts formed from photoreaction of tryptophan with other trihalo and monohalocompounds are also determined. The indole ring of tryptophan can be modified with various additions from halocompounds, including the addition of labels to the indole ring via methylene groups. This opens possibilities for generating novel tryptophan based fluorophores and protein labeling strategies using this photochemistry.

A Rapid and Fluorogenic TMP-AcBOPDIPY Probe for Covalent Labeling of Proteins in Live Cells

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

Resin supported acyl carrier protein labeling strategies.

The post-translational modifying enzymes phophopantetheinyl transferase and acyl carrier protein hydrolase have shown utility in the functional modification of acyl carrier proteins. Here we develop these tools as immobilized biocatalysts on agarose supports. New utility is imparted through these methods, enabling rapid and label-independent protein purification. Immobilization of acyl carrier protein is also demonstrated for rapid activity assays of these 4'-phosophopantetheine modifying enzymes, displaying a particular advantage in the case of phosphopantetheine removal, where few orthogonal techniques have been demonstrated. These tools further enrich the suite of functional utility of 4'-phosophopantetheine chemistry, with applications to protein functionalization, materials, and natural product biosynthetic studies.

Aqueous oxidative Heck reaction as a protein-labeling strategy.

An increasing number of chemical reactions are being employed for bio-orthogonal ligation of detection labels to protein-bound functional groups. Several of these strategies, however, are limited in their application to pure proteins and are ineffective in complex biological samples such as cell lysates. Here we present the palladium-catalyzed oxidative Heck reaction as a new and robust bio-orthogonal strategy for linking functionalized arylboronic acids to protein-bound alkenes in high yields and with excellent chemoselectivity even in the presence of complex protein mixtures from living cells. Advantageously, this reaction proceeds under aerobic conditions, whereas most other metal-catalyzed reactions require inert atmosphere.

Marked for depth

An in vivo protein-labeling strategy could enable more comprehensive surveys of organelle proteomic contents.

Superresolution imaging of HIV in infected cells with FlAsH-PALM.

Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffraction-limited resolution of conventional light microscopy (∼200-300nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.

One- and Two-Photon Live Cell Imaging Using a Mutant SNAP-Tag Protein and Its FRET Substrate Pairs

A small molecule-assisted protein labeling strategy based on a mutant SNAP-Tag (mSNAP) and its FRET substrate pairs has been developed. Both one- and two-photon fluorescence microscopic experiments were successfully demonstrated in living cells.

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.

Development of a new protein labeling strategy, oxidation labeling. part 1: Preliminary evaluation and synthesis of tautomycin containing a metal coordinating unit

In the current work we present our preliminary evaluation of a new protein labeling strategy, namely oxidation labeling. We found that a bis(2-picolyl) amine analogue coordinating Cu + was able to oxidize histidine to oxo-histedine in a small peptide by generating reactive oxygen species upon exposure to hydrogen peroxide. The bis(2-picolyl) amine unit was then incorporated into the natural product tautomycin via an oxime linker. The compound, which showed good activity toward protein phosphatase 1γ (PP1γ), will be used in oxidation labeling studies with PP1γ.

Methyl groups as probes of supra-molecular structure, dynamics and function

The development of new protein labeling strategies, along with optimized experiments that exploit the label, have significantly impacted on the types of biochemical problems that can now be addressed by solution NMR spectroscopy. Here we describe how methyl labeling of key residues in a highly deuterated protein background has facilitated studies of the structure, dynamics and interactions of supra-molecular particles. The methyl-labeling approach is briefly reviewed, followed by a summary of applications to three different molecular machines so as to illustrate the types of questions that can now be addressed. Areas where future innovations will lead to yet further improvements are highlighted as well.

Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.

Selective labeling of selenomethionine residues in proteins with a fluorescent derivative of benzyl bromide

Benzyl bromide is used as a reagent for the selective modification of methionine residues in proteins. We here explored the suitability of the bromobenzyl moiety as a reactive group for the targeted fluorescent labeling of methionine and selenomethionine residues in proteins. A novel labeling reagent ( N, N′, N′-trimethyl- N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- N′-( p-bromomethylbenzyl)-ethylenediamine, NBD-BBr) was synthesized and tested for reactivity with two model proteins containing single methionine or selenomethionine residues. The amounts of reagent and reactions times required for modification of methionine resulted in side reactions with other amino acid residues, a finding which was also confirmed for benzyl bromide itself. However, with selenomethionine, lower concentrations and shorter reaction times were sufficient for NBD-BBr modification. Under these conditions, labeling was confined to selenomethionine residues with one but not the other model protein. Where applicable, the protein labeling strategy characterized here is rapid and efficient. It should be useful in combination with cysteine-specific labeling if dual site-specific modification is desired.