Christine Koehler

Amino Acids for Diels–Alder Reactions in Living Cells

The endeavour to perform tailored chemical reactions in the challenging environment of the intact cell delves deeply into the biological sciences. Requirements include strict bioorthogonality of the reactants and reactions that occur spontaneously and quickly in an aqueous environment or at the interface of membranes. Commonly used reactions that meet these criteria are Staudinger ligations and various forms of click chemistry. The most prominent among the latter is the Huisgen-type [3+2] cycloaddition between azides and alkynes. 2] Through the seminal work of the Bertozzi group, this reaction was stripped of its need for Cu catalysis by straining the alkyne group, thereby making this chemistry (termed strain-promoted alkyne–azide chemistry, SPAAC) viable in intact cells as well as in living animals. These reactions have been widely used to label molecules on cell surfaces and, in a few cases, inside the cell, for instance to label lipids, nucleotides, or carbohydrates. Another exciting click variant is strain-promoted inverse-electrondemand Diels–Alder cycloaddition (SPIEDAC), which can exhibit accelerated reaction rates by using strained reactants and furthermore is irreversible because of the loss of N2 (Scheme 1). This chemistry has been used in cells to label small molecules and is magnitudes faster than the classical Huisgen-type cycloadditions. To date, most biological applications of SPAAC or SPIEDAC do not involve modifications of proteins but instead alter cellular molecules that are not genetically encoded, such as metabolically incorporated sugars. Current tools for site-specific labeling of proteins within the cell use fluorescent protein fusions, self-alkylating protein additions, or high-affinity binding domains. The smallest size of artificial protein modifications currently available to introduce fluorescent labels are tetracysteine motifs consisting of six amino acids. Ideally the modification unit would be only a single artificial amino acid suitable for specific chemistry in cells. The introduction of such unnatural amino acids (UAAs) is possible by codon reassignment or by suppression of the Amber stop codon. For fluorescent labeling, genetically encoded azides can be used, but azides typically suffer from intracellular reduction. Furthermore, encoding the azide jeopardizes the design of a fluorogenic labeling scheme. 16] Fluorogenicity is of particular relevance for high-contrast imaging and super-resolution techniques, since dyes are turned on only after successful labeling, while nonspecifically attached dyes remain quenched. Rather than encoding azides, a more suitable approach is the use of an amino acid that carries the strained reactant, that is, a cyclooctyne group, thereby leaving the nitrogen-bearing reactants to serve as part of a fluorogenic probe. If suppression of the Amber stop codon is used, a single residue in a specified protein can then be replaced with the strained alkyne. This type of protein labeling by using an artificially introduced cyclooctyne amino acid and fluorogenic azides Scheme 1. a) Structures of strained alkene and alkyne UAAs. b) Reaction scheme showing orthogonality and cross-reactivity of SPIEDAC and SPAAC with fluorogenic tetrazine-functionalized dyes (gray sphere) and azide-functionalized dyes (green sphere). Dyes coupled to tetrazine are only fluorescent (green) after successful labeling.

Genetically Encoded Copper-Free Click Chemistry

The ability to visualize biomolecules within living specimen by engineered fluorescence tags has become a major tool in modern biotechnology and cell biology. Encoding fusion proteins with comparatively large fluorescent proteins (FPs) as originally developed by the Chalfie and Tsien groups is currently the most widely applied technique.[1] As synthetic dyes typically offer better photophysical properties than FPs, alternative strategies have been developed based on genetically encoding unique tags such as Halo and SNAP tags, which offer high specificity but are still fairly large.[2] Small tags like multi-histidine[3] or multi-cysteine motifs[4] may be used to recognize smaller fluorophores, but within the cellular environment they frequently suffer from poor specificity as their basic recognition element is built from native amino acid side chains. Such drawbacks may be overcome by utilizing bioorthogonal chemistry that relies on coupling exogenous moieties of non-biological origin under mild physiological conditions. A powerful chemistry that fulfils these requirements is the Huisgen type (3+2) cycloaddition between azides and alkynes (a form of click chemistry[5]). By utilizing supplementation-based incorporation techniques and click reactions Beatty et al. coupled azide derivatized dyes to Escherichia coli expressing proteins bearing linear alkynes.[6] However, this azide–alkyne cycloaddition required copper(I) as a catalyst (CuAAC), which strongly reduces biocompatibility (but see Ref. [7]). This limitation has been overcome by Bertozzi and co-workers, who showed that the “click” reaction readily proceeds when utilizing ring-strained alkynes as a substrate[8] and since then this strain-promoted azide–alkyne cycloaddition (SPAAC) has found increasing applications in labeling, for example, carbohydrates,[9] nucleotides,[10] and lipids.[11] Further expanding the potential of this approach, Ting and co-workers engineered a lipolic acid ligase which ligates a small genetically encoded recognition peptide to a cylcooctyne-containing substrate. In a second step the incorporated cyclooctyne moiety then functioned as a specific site for labeling in cells.[12]

Plasticity of an Ultrafast Interaction between Nucleoporins and Nuclear Transport Receptors

Summary The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.

Click strategies for single-molecule protein fluorescence.

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.

Facilitated aggregation of FG nucleoporins under molecular crowding conditions.

Intrinsically disordered and phenylalanine–glycine‐rich nucleoporins (FG Nups) form a crowded and selective transport conduit inside the NPC that can only be transited with the help of nuclear transport receptors (NTRs). It has been shown in vitro that FG Nups can assemble into two distinct appearances, amyloids and hydrogels. If and how these phenomena are linked and if they have a physiological role still remains unclear. Using a variety of high‐resolution fluorescence and electron microscopic (EM) tools, we reveal that crowding conditions mimicking the NPC environment can accelerate the aggregation and amyloid formation speed of yeast and human FG Nups by orders of magnitude. Aggregation can be inhibited by NTRs, providing a rationale on how the cell might control amyloid formation of FG Nups. The superb spatial resolving power of EM also reveals that hydrogels are enlaced amyloid fibres, and these findings have implications for existing transport models and for NPC assembly.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements

Significance Conformational properties of unfolded and intrinsically disordered proteins (IDPs) under native conditions are important for understanding the details of protein folding and the functions of IDPs. The average dimensions of these systems are quantified using the mean radius of gyration and mean end-to-end distance, measured by small-angle X-ray scattering (SAXS) and single-molecule Förster resonance energy transfer (smFRET), respectively, although systematic discrepancies emerge from these measurements. Through holistic sets of studies, we find that the disagreements arise from chemical heterogeneity that is inherent to heteropolymeric systems. This engenders a decoupling between different measures of overall sizes and shapes, thus leading to discrepant inferences based on SAXS vs. smFRET. Our findings point the way forward to obtaining comprehensive descriptions of ensembles of heterogeneous systems. Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE. For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE, whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

Highly Stable trans‐Cyclooctene Amino Acids for Live‐Cell Labeling

trans-Cyclooctene groups incorporated into proteins via non-canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chemistry. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans-cyclooct-2-ene isomers (1 a,b). We further show that the axially connected isomer has a half-life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as determined by stopped-flow experiments. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.

Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range

Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.

Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy.

Abstract Super‐resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine‐based GCE system for click chemistry, combined with DNA‐PAINT microscopy, enables the visualization of even low‐abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue‐specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.

Bisazide Cyanine Dyes as Fluorogenic Probes for Bis-Cyclooctynylated Peptide Tags and as Fluorogenic Cross-Linkers of Cyclooctynylated Proteins

Herein we present the synthesis and fluorogenic characterization of a series of double-quenched bisazide cyanine probes with emission maxima between 565 and 580 nm that can participate in covalent, two-point binding bioorthogonal tagging schemes in combination with bis-cyclooctynylated peptides. Compared to other fluorogenic cyanines, these double-quenched systems showed remarkable fluorescence intensity increase upon formation of cyclic dye-peptide conjugates. Furthermore, we also demonstrated that these bisazides are useful fluorogenic cross-linking platforms that are able to form a covalent linkage between monocyclooctynylated proteins.