Sub-stoichiometric Labeling Research Articles

FISIK: Framework for the Inference of In Situ Interaction Kinetics from Single-Molecule Imaging Data

Recent experimental and computational developments have been pushing the limits of live-cell single-molecule imaging, enabling the monitoring of intermolecular interactions in their native environment with high spatiotemporal resolution. However, interactions are captured only for the labeled subset of molecules, which tends to be a small fraction. As a result, it has remained a challenge to calculate molecular interaction kinetics, in particular association rates, from live-cell single-molecule tracking data. To overcome this challenge, we developed a mathematical modeling-based Framework for the Inference of in Situ Interaction Kinetics (FISIK) from single-molecule imaging data with substoichiometric labeling. FISIK consists of (I) devising a mathematical model of molecular movement and interactions, mimicking the biological system and data-acquisition setup, and (II) estimating the unknown model parameters, including molecular association and dissociation rates, by fitting the model to experimental single-molecule data. Due to the stochastic nature of the model and data, we adapted the method of indirect inference for model calibration. We validated FISIK using a series of tests in which we simulated trajectories of diffusing molecules that interact with each other, considering a wide range of model parameters, and including resolution limitations, tracking errors, and mismatches between the model and the biological system it mimics. We found that FISIK has the sensitivity to determine association and dissociation rates, with accuracy and precision depending on the labeled fraction of molecules and the extent of molecule tracking errors. For cases where the labeled fraction is too low (e.g., to afford accurate tracking), combining dynamic but sparse single-molecule imaging data with almost-whole population oligomer distribution data improves FISIK’s performance. All in all, FISIK is a promising approach for the derivation of molecular interaction kinetics in their native environment from single-molecule imaging data with substoichiometric labeling.

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15-27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase.

Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ∼0.1 s-1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.

Single Molecule Tracking and Localization of Mitochondrial Protein Complexes in Live Cells.

Mitochondria are the power plant of most non-green eukaryotic cells. An understanding of their function and regulation is only possible with the knowledge of the spatiotemporal dynamics of their proteins. Mitochondrial membrane proteins involved in diverse functions like protein import, cell respiration, metabolite transport, and mitochondrial morphology are mobile within membranes. Here, we provide a protocol for a superresolution fluorescence microscopy technique named tracking and localization microscopy (TALM) that allows for localization and diffusion analysis of single mitochondrial membrane proteins in situ in cell cultures. This noninvasive imaging technique is a useful tool to reveal the spatiotemporal organization of proteins in diverse mitochondrial membrane compartments in living cells. Proteins of interest are tagged with the HaloTag® and specifically labeled with functionalized rhodamine dyes. The method profits from low abundance of proteins and therefore works better with substoichiometric labeling of HaloTag®-tagged proteins. In particular, the use of photostable bright rhodamine dyes enables the specific tagging and localization of single molecules with a calculated precision below 20 nm and the recording of single trajectories.

Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid.

Bioorthogonal reactions for labeling biomolecules in live cells have been limited by slow reaction rates or low component selectivity and stability. Ideal bioorthogonal reactions with high reaction rates, high selectivity, and high stability would allow for stoichiometric labeling of biomolecules in minutes and eliminate the need to wash out excess labeling reagent. Currently, no general method exists for controlled stoichiometric or substoichiometric labeling of proteins in live cells. To overcome this limitation, we developed a significantly improved tetrazine-containing amino acid (Tet-v2.0) and genetically encoded Tet-v2.0 with an evolved aminoacyl-tRNA synthetase/tRNA(CUA) pair. We demonstrated in cellulo that protein containing Tet-v2.0 reacts selectively with cyclopropane-fused trans-cyclooctene (sTCO) with a bimolecular rate constant of 72,500 ± 1660 M(-1) s(-1) without reacting with other cellular components. This bioorthogonal ligation of Tet-v2.0-protein reacts in cellulo with substoichiometric amounts of sTCO-label fast enough to remove the labeling reagent from media in minutes, thereby eliminating the need to wash out label. This ideal bioorthogonal reaction will enable the monitoring of a larger window of cellular processes in real time.

150 Getting a grip on alpha-synuclein amyloid oligomers

Misfolding and aggregation of proteins into nanometer-scale fibrillar assemblies is a hallmark of many neurodegenerative diseases. Aggregation of the human alpha-synuclein protein is implicated in the etiology of Parkinson’s disease. A particularly relevant question is the role of early oligomeric aggregates of alpha-synuclein in modulating the dynamics of protein aggregation, and in the interactions with essential cellular components. However, very little is known about the molecular details of these aggregate species. For large protein aggregates, such as alpha-synuclein oligomers, it is very difficult to determine the number of monomers that form an oligomer using conventional techniques. We have developed a method that uses sub-stoichiometric labeling, that is, only a fraction of the monomers contains a fluorescent label, in combination with single-molecule photobleaching to determine the number of monomers per oligomer (Zijlstra et al., 2012). The number of bleaching steps gives the number of fluorescent labels per oligomer. Knowing the exact label density, that is, the fraction of labeled monomers at the start of the aggregation, we can correlate the number of fluorescent labels per oligomer to the total number of monomers. Using this method, we can determine the composition, probe the distribution in the number of monomers per oligomer, and investigate the influence of the fluorescent label on the aggregation process. For wild-type alpha-synuclein, we find no distribution in the number of monomers per oligomer and find a single, well-defined oligomeric species consisting of ∼30 monomers per oligomer. On the other hand, for oligomers formed in the presence of dopamine, we find a distinctly bimodal distribution suggesting the existence of two populations of oligomeric species.

Cover Picture: Molecular Composition of Sub-stoichiometrically Labeled α-Synuclein Oligomers Determined by Single-Molecule Photobleaching (Angew. Chem. Int. Ed. 35/2012)

In amyloid diseases, monomeric proteins self-assemble into supramolecular aggregates. In their Communication on page 8821 ff. V. Subramaniam et al. describe a new method that combines single-molecule photobleaching approaches with sub-stoichiometric labeling with fluorophores to determine the molecular composition of these large protein aggregates. The cover picture shows the aggregation of combinations of labeled and unlabeled monomers and the subsequent photobleaching analysis of single aggregates.

Molecular Composition of Sub‐stoichiometrically Labeled α‐Synuclein Oligomers Determined by Single‐Molecule Photobleaching

Bleaching proteins: Single-molecule photobleaching approaches and sub-stoichiometric labeling with fluorophores give insight into the number of monomers that form a specific α-synuclein oligomer. The results show that this α-synuclein oligomer is present as a single, well-defined species consisting of 31 monomers.

Establishing the Composition of Alpha-Synuclein Oligomers using Single-Molecule Photobleaching

Growing evidence suggests that oligomeric aggregation intermediates of the human alpha-synuclein protein are toxic to neurons in Parkinson's disease. Very little is known about the molecular details of these oligomeric aggregates. Conventional techniques to determine the number of monomers per oligomer, such as size exclusion chromatography or mass spectrometry, give estimates ranging from less than 10 to more than 60. It is unclear if this spread is due to influences from the technique used or if it reflects size heterogeneity of the oligomers.Single-molecule photobleaching allows for direct probing of the number of labeled monomers per oligomer, but is not typically applicable for large oligomers (>10 monomers). To overcome this problem, we develop a method that uses sub-stoichiometric labeling, that is, only a fraction of the monomers contain a fluorescent label, in combination with single-molecule photobleaching. The number of bleaching steps now gives the number of fluorescent labels instead of the number of monomers. Via the label probability mass function we link the number of fluorescent labels to the total number of monomers.Using this approach, we find a single, well defined alpha-synuclein oligomer consisting of 31 monomers. We also demonstrate that the aggregation is stochastic and that there is no influence of the fluorescent label on the aggregation. Fluorescence correlation spectroscopy data indicate a very loose packing of the oligomers. In combination with tryptophan fluorescence measurements, we propose a structural model for the oligomers.

Mechanism-Based Enzyme Inactivation Using an Allyl Sulfoxide-Allyl Sulfenate Ester Rearrangement1.

2-Amino-4-chloro-5-(p-nitrophenylsulfinyl)pentanoic acid (1) has been synthesized and shown to induce mechanism-based inactivation of two pyridoxal phosphate dependent enzymes: (1) cystathionine γ-synthetase, which catalyzes a γ-replacement reaction in bacterial methionine biosynthesis; and (2) methionine γ-lyase, which catalyzes a γ-elimination reaction in bacterial methionine breakdown. The inactivations are irreversible and display saturation kinetics. Each enzyme incorporates roughly 1 mol of tritium per mol of enzyme monomer when inactivated by 2-amino-4-chloro-5-(p-nitro[3H]phenyl-sulfinyl)pentanoic acid (la), confirming that the modification of each protein is covalent and stoichiometric. Substoichiometric labeling (0.12 mol of tritium per mol of enzyme monomer) is given when methionine γ-lyase is fully inactivated by 2-amino-4-chloro-5-[3H]-5-p-nitrophenylsulfinyl)pentanoic acid (lb). Both enzymes, inactivated by 1, are susceptible to reactivation by thiols. Inactivated cystathionine γ-synthetase recovers 25% of its catalytic activity upon incubation with excess dithiothreitol, while methinonine γ-lyase is 100% reactivated by dithiothreitol, mercaptoethanol, and mercaptopropionate. Reactivation generates p-nitrophenylthiolate anion, which forms, in the case of methionine γ-lyase, stoichiometrically with enzyme reactivated. Both enzymes are "protected" from inactivation by 1 in the presence of thiols, which simultaneously generates p-nitrophenylthiol. In the presence of dithiothreitol, the protection reaction gives p-nitrophenylthiol production with pseudo-first-order kinetics. 2-Amino-4-chloro-5-(p-tolylsulfinyl)pentanoic acid (2) and 2-amino-4-(p-nitrophenylsulfinyl)-5-chloropentanoic acid (3), the reverse regioisomer of 1, have also been prepared and give no evidence of inactivation of either enzyme. The data are taken to indicate a novel form of suicide inactivation (Scheme II) wherein β-carbanion-assisted γ-halide elimination generates an allyl sulfoxide-enzyme-pyridoxal adduct (4) which undergoes spontaneous 2,3-sigmatropic rearrangement to an electrophilic allyl sulfenate ester (5). The latter is then captured by an enzymic nucleophile to give an inactive enzyme 6, which may be a mixed disulfide or, less likely, a sulfenamide.