Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors.

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.

Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET.

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells.

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels-Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to ß-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo ß-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.

Inducible Genetic Code Expansion in Eukaryotes.

Genetic code expansion (GCE) is a versatile tool to site-specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl-tRNA-synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T-REx or Tet-On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T-REx system enables simultaneous control of tRNA and RS expression.

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

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.

Genetic code expansion for multiprotein complex engineering.

We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.

Bistetrazine-Cyanines as Double-Clicking Fluorogenic Two-Point Binder or Crosslinker Probes.

Fluorogenic probes can be used to minimize the background fluorescence of unreacted and nonspecifically adsorbed reagents. The preceding years have brought substantial developments in the design and synthesis of bioorthogonally applicable fluorogenic systems mainly based on the quenching effects of azide and tetrazine moieties. The modulation power exerted by these bioorthogonal motifs typically becomes less efficient on more conjugated systems; that is, on probes with redshifted emission wavelength. To reach efficient quenching, that is, fluorogenicity, even in the red range of the spectrum, we present the synthesis, fluorogenic, and conjugation characterization of bistetrazine-cyanine probes with emission maxima between 600 and 620 nm. The probes can bind to genetically altered proteins harboring an 11-amino acid peptide tag with two appending cyclooctyne motifs. Moreover, we also demonstrate the use of these bistetrazines as fluorogenic, covalent cross-linkers between monocyclooctynylated proteins.

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.

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

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.

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.

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.

Quaternary structure of the yeast Arc1p-aminoacyl-tRNA synthetase complex in solution and its compaction upon binding of tRNAs.

In the yeast Saccharomyces cerevisiae, the aminoacyl-tRNA synthetases (aaRS) GluRS and MetRS form a complex with the auxiliary protein cofactor Arc1p. The latter binds the N-terminal domains of both synthetases increasing their affinity for the transfer-RNA (tRNA) substrates tRNA(Met) and tRNA(Glu). Until now, structural information was available only on the enzymatic domains of the individual aaRSs but not on their complexes with associated cofactors. We have analysed the yeast Arc1p-complexes in solution by small-angle X-ray scattering (SAXS). The ternary complex of MetRS and GluRS with Arc1p, displays a peculiar extended star-like shape, implying possible flexibility of the complex. We reconstituted in vitro a pentameric complex and demonstrated by electrophoretic mobility shift assay that the complex is active and contains tRNA(Met) and tRNA(Glu), in addition to the three protein partners. SAXS reveals that binding of the tRNAs leads to a dramatic compaction of the pentameric complex compared to the ternary one. A hybrid low-resolution model of the pentameric complex is constructed rationalizing the compaction effect by the interactions of negatively charged tRNA backbones with the positively charged tRNA-binding domains of the synthetases.

Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells.

Genetic code expansion (GCE) reprograms the translational machinery to site-specifically incorporate noncanonical amino acids (ncAAs) into a selected protein. The efficiency of GCE in mammalian cells might be compromised by cellular stress responses, among which, the protein kinase R(PKR)-dependent eIF2α phosphorylation pathway can reduce translation rates. Here we test several strategies to engineer the eIF2α pathway and boost the rate of translation and show that such interventions increase GCE efficiency in mammalian cells. In particular, addition of the N-terminal PKR fragment (1-174) provides a substantial enhancement in cytoplasmic GCE and also in GCE realized by OTOs (orthogonally translating designer organelles), which built on the principle of 2D phase separation to enable mRNA-selective ncAA incorporation. Our study demonstrates an approach for improving the efficiency of GCE and provides a means by which the power of designer organelles can be further optimized to tune protein translation.

Genetic Code Expansion for Site-Specific Labeling of Antibodies with Radioisotopes.

Due to their target specificity, antibody-drug conjugates─monoclonal antibodies conjugated to a cytotoxic moiety─are efficient therapeutics that can kill malignant cells overexpressing a target gene. Linking an antibody with radioisotopes (radioimmunoconjugates) enables powerful diagnostics and/or closely related therapeutic applications, depending on the isotope. To generate site-specific radioimmunoconjugates, we utilized genetic code expansion and subsequent conjugation by inverse electron-demand Diels-Alder cycloaddition reactions. We show that, using this approach, site-specific labeling of trastuzumab with either zirconium-89 (89Zr) for diagnostics or lutetium-177 (177Lu) for therapeutics yields efficient radioimmunoconjugates. Positron emission tomography imaging revealed a high accumulation of site-specifically 89Zr-labeled trastuzumab in tumors after 24 h and low accumulation in other organs. The corresponding 177Lu-trastuzumab radioimmunoconjugates were comparably distributed in vivo.

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.

MultiBacTAG-Genetic Code Expansion Using the Baculovirus Expression System in Sf21 Cells.

The combination of genetic code expansion (GCE) and baculovirus-based protein expression in Spodoptera frugiperda cells is a powerful tool to express multiprotein complexes with site-specifically introduced noncanonical amino acids. This protocol describes the integration of synthetase and tRNA gene indispensable for GCE into the backbone of the Bacmid, the Tn7-mediated transposition of various genes of interest, as well as the final expression of protein using the MultiBacTAG system with different noncanonical amino acids.

Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID.

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

Enamel fluoride retention after application of fluoride-containing rubber cups.

PURPOSE: To compare the fluoride retention in bovine enamel after using fluoride-containing rubber cups or a conventional rubber cup in combination with a fluoride-containing polishing paste. MATERIALS AND METHODS: From each of 20 bovine incisors, 5 enamel slabs with a diameter of 3 mm were sectioned and assigned to 5 groups (A,B,C,D,E). Group A was left untreated and served as the negative control. Group B was polished with a rubber cup combined with a fluoride-containing prophylaxis paste for 10 s (Hawe Cleanic). Groups C and D were treated in the same manner by using a fluoride-containing rubber cup with 2,262 ppm or 9,048 ppm fluoride as NaF. Group E enamel was brushed for 10 s with a toothbrush and a slurry of distilled water and fluoride gel with 12,500 ppm fluoride as NaF and AmF (Elmex Gelee) and served as the positive control. Th e specimens were analyzed using the method of Caslavska et al. After determining the KOH soluble fluoride on the enamel surface, the content of structurally bound fluoride was analyzed in three consecutive layers of 30 microm. ANOVA with Bonferroni correction was used for statistical analysis (P< 0.05). RESULTS: For the KOH-soluble fluoride, Groups B and D accumulated more KOH-soluble fluoride than the negative control and less than the positive control. With respect to the structurally bound fluoride, the accumulation in Group D was higher when compared to all other groups. It was concluded that a rubber cup with 9,048 ppm fluoride may be an effective alternative to the use of a fluoride-containing prophylaxis paste.