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In his commentary in this issue of Molecular Cell , 1 Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications. In his commentary in this issue of Molecular Cell ,1 Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Summary of immense potential of genetic code expansion in eukaryotes • Discussion of strategies for mitigating limitations of genetic code expansion • Pitfalls of genetic code expansion enabled in vivo click-labelling with fluorophores Following decades of innovation and perfecting, genetic code expansion has become a powerful tool for in vivo protein modification. Some of the major hurdles that had to be overcome include suboptimal performance of GCE-specific translational components in host systems, competing cellular processes, unspecific modification of the host proteome and limited availability of codons for reassignment. Although strategies have been developed to overcome these challenges, there is critical need for further advances. Here we discuss the current state-of-the-art in genetic code expansion technology and the issues that still need to be addressed to unleash the full potential of this method in eukaryotic cells. [ABSTRACT FROM AUTHOR]

The compartmentalization of specific functions into specialized organelles is a key feature of eukaryotic life. In particular, dynamic biomolecular condensates that are not membrane enclosed offer exciting opportunities for synthetic biology. In recent years, multiple approaches to generate and control condensates have been reported. Notably, multiple orthogonally translating organelles were designed that enable precise protein engineering inside living cells. Despite being built from only very few components, orthogonal translation can be engineered with subresolution precision at different places inside the same cell to create mammalian cells with multiple expanded genetic codes. This provides a pathway to engineer multiple proteins with multiple and distinct functionalities inside living eukaryotes and provides a general strategy toward spatially orthogonal enzyme engineering. [Display omitted] • Synthetic organelle formation can be controlled with small molecules or light. • Synthetic membraneless organelles enable mRNA selective genetic code expansion. • Two-dimensional phase separation creates orthogonal enzymes by subresolution spatial separation. • Film-like organelles generate cells with two expanded genetic codes. [ABSTRACT FROM AUTHOR]

The nuclear pore complex (NPC) forms a permeability barrier between the nucleus and the cytoplasm. Molecules that are able to cross this permeability barrier encounter different disordered phenylalanine glycine rich nucleoporins (FG-Nups) that act as a molecular filter and regulate the selective NPC crossing of biomolecules. In this review, we provide a current overview regarding the interaction mechanism between FG-Nups and the carrier molecules that recognize and enable the transport of cargoes through the NPC aiming to understand the general molecular mechanisms that facilitate the nucleocytoplasmic transport. [ABSTRACT FROM AUTHOR]

Total internal reflection fluorescence microscopy (TIRFM) can offer favorably high signal-to-noise observation of biological mechanisms. TIRFM can be used routinely to observe even single fluorescent molecules for a long duration (several seconds) at millisecond time resolution. However, to keep the investigated sample in the evanescent field, chemical surface immobilization techniques typically need to be implemented. In this review, we describe some of the recently developed novel nanodevices that overcome this limitation enabling long-term observation of free single molecules and outline their biological applications. The working concept of many devices is compatible with high-throughput strategies, which will further help to establish unbiased single molecule observation as a routine tool in biology to study the molecular underpinnings of even the most complex biological mechanisms. [ABSTRACT FROM AUTHOR]

An evolutionary advantage of intrinsically disordered proteins (IDPs) is their ability to bind a variety of folded proteins—a paradigm that is central to the nucleocytoplasmic transport mechanism, in which nuclear transport receptors mediate the translocation of various cargo through the nuclear pore complex by binding disordered phenylalanine–glycine-rich nucleoporins (FG-Nups). FG-Nups are highly dynamic, which poses a substantial problem when trying to determine precisely their function using common experimental approaches. FG-Nups have been studied under a variety of conditions, ranging from those that constitute single-molecule measurements to physiological concentrations at which they can form supramolecular structures. In this review, I describe the physicochemical properties of FG-Nups and compare them to those of other disordered systems, including well-studied IDPs. From this comparison, it is apparent that FG-Nups not only share some properties with IDPs in general but also possess unique characteristics that might be key to their central role in the nucleocytoplasmic transport machinery. [ABSTRACT FROM AUTHOR]

Genetic code expansion is emerging as an important tool for manipulation and labeling of proteins in vitro and in vivo . In combination with click-chemistry it allows site-specific labeling of target proteins with small organic fluorophores. This is achieved by cotranslational incorporation of noncanonical amino acids (ncAAs) in target proteins via orthogonal tRNA/amino-acyl tRNA synthetase pairs. In a subsequent step, ncAAs are labeled with small dyes via click-chemistry. Small labeling tags and free choice of which fluorophore to use and where to put it into the protein are of particular importance for single molecule science and superresolution microscopy. Such genetically encoded click chemistry tools facilitate high resolution studies of protein function in live cells, viral imaging, as well as the improved design of antibody-drug conjugates. [ABSTRACT FROM AUTHOR]

Contemporary structural biology research promises more than just static snap-shots of molecular machineries. This goal is not just facilitated by combining different structural biology techniques, but also by new tools from the field of protein and genetic engineering, as well as from chemistry. Genetic encoding of noncanonical amino acids (ncAAs) through codon-suppression technology provides an excellent opportunity to probe biomolecules using different structural biology methods. In this article, we review the applications of ncAA incorporation into proteins for determining structural information through various techniques with the main focus on crosslinking mass spectrometry and single-molecule FRET-based techniques. Furthermore, advances and limitations of the incorporation of multiple ncAAs are discussed, with respect to design of an ideal host organism for modern and integrative structural biology research. [ABSTRACT FROM AUTHOR]

Phase separation of proteins is recognized as an important aspect of cellular organization and disease mechanisms. Shin et al. introduce a novel optogenetic tool, which enables different phase-space regimes inside living cells to be assessed and the transition paths between them to be studied with unprecedented spatiotemporal control. [ABSTRACT FROM AUTHOR]

Intrinsisch ungeordnete Proteine (IUPs) können unterschiedliche Interaktionspartner binden. Mehrere Bindungsregionen in IUPs, die durch komplexe kooperative Effekte zusammenwirken, vereinfachen solche Interaktionen. Gleichzeitig erschweren sie aber die Analyse solcher IUP-Komplexe. Um diese Schwierigkeit anzugehen, kombinierten wir Fluoreszenzkorrelationsspektroskopie mit zeitaufgelöster Polarisationsspektroskopie, um die Eigenschaften der Bindung des IUP Nucleoporin 153 (Nup153) mit Transportrezeptoren (TRs) des Kerntransports zu untersuchen. Die Detektion von flexibler Segmentbewegung im Rückgrat des Proteins Nup153 zeigte lokale, regionsspezifische Bindungspräferenzen von TRs. Solche Eigenschaften bleiben oft verborgen, wenn allgemeine - nicht regionsspezifische - Eigenschaften des IUP untersucht werden. Die Affinität von funktionell und strukturell unterschiedlichen TRs für bestimmte Regionen von Nup153 können sich um Größenordnungen unterscheiden - ein Befund mit Bedeutung für verschiedene Transportwege im Kerntransport. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time-resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region-specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes-a result with implications for the diversity of transport routes in nucleocytoplasmic transport. [ABSTRACT FROM AUTHOR]

I109 --> 254.3 --> The mechanisms by which intrinsically disordered proteins (IDPs) engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function. Nuclear transport receptors (NTRs) can move through the central channel of the NPC which is filled with hundreds of phenylalanine‐glycine‐rich nucleoporins (FG‐Nups) reaching millimolar concentrations with elusive conformational plasticity. Since site‐specific labeling of proteins with small but highly photostable fluorescent dyes inside cells remains the major bottleneck for directly studying protein dynamics in the cellular interior, we have now developed a semi‐synthetic strategy based on novel artificial amino acids that are easily and site‐specifically introduced into any protein by the natural machinery of the living cell via a newly developed thin‐film synthetic organelle that equips the living cell with up to three genetic codes. This allowed us to develop an experimental approach combining site‐specific fluorescent labeling of IDPs in non‐fixed cells with fluorescent lifetime imaging microscopy (FLIM) to directly decipher the plasticity of FG‐Nups via FRET. Our study enabled a conformational look on the condensated IDPs in the sub‐resolution (roughly (50 nm)3 small cavity) cavity of the NPC. By measuring the end‐to‐end distances of different segments of the labeled FG‐Nups using FLIM‐FRET, we can extract the scaling exponent, which directly describes the conformations of FG‐Nups at their functional status as well as the solvent quality in the cellular and even inner NPC environment. Reinkemeier CD, Lemke EA. Dual film‐like organelles enable spatial separation of orthogonal eukaryotic translation. Cell. 2021 Sep 16;184(19):4886‐4903.e21 Celetti G, Paci G, Caria J, VanDelinder V, Bachand G, Lemke EA. The liquid state of FG‐nucleoporins mimics permeability barrier properties of nuclear pore complexes. J Cell Bio. (2020) Jan 6;219(1). Reinkemeier CD, Estrada Girona G, Lemke EA, 2019 Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Science, Mar 29;363(6434) Nikić I, Estrada Girona G, Kang JH, Paci G, rei S, Koehler C, Shymanska NV, Ventura Santos C, Spitz D, Lemke EA, Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy. Angew Chem Int Ed Engl. (2016) Dec 23;55(52):16172‐16176 [ABSTRACT FROM AUTHOR]

[Display omitted] • Microtubule-based synthetic organelles enable orthogonal translation of selected mRNAs. • Synthetic fiber-like organelles coat microtubules. • A eukaryotic cell with two genetic codes. • Recruited mRNAs get translated selectively with an expanded genetic code. Membraneless organelles are capable of selectively performing complex tasks in living cells despite dynamically exchanging with their surroundings. This is an exquisite example how self-organization of proteins and RNAs can lead to more complex functionalities in living systems. Importantly, the absence of a membrane boundary can enable easier access to larger macromolecular complexes that can be challenging to be transported across a membrane. We previously formed orthogonally translating designer membraneless organelles by combining phase separation with kinesin motor proteins to highly enrich engineered translational factors in large organelles. We also showed that even submicron thick designer organelles can be formed, by mounting them onto membranes, which, presumable assisted by 2D condensation, leads to thin film-like condensates. In this study we show that orthogonal translation can also be built with fiber-like appearing organelles. Here, the microtubule-end binding protein EB1 was used to form fiber-like OT organelles along the microtubule cytoskeleton that perform highly selective and efficient orthogonal translation. We also show an improved simplified design of OT organelles. Together this extends OT organelle technology and demonstrates that the microtubule cytoskeleton is a powerful platform for advanced synthetic organelle engineering. [ABSTRACT FROM AUTHOR]

While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard- and software developments, but also by the availability and suitability of protein-engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The author discuss options for designing fluorescent sensors and reporters and strategies to develop their properties. They define sensor and reporter molecules as related to cell biology and note that bacterial and plant proteins and protein domains that lack enzyme activity can be useful in building sensors in mammalian cells or intramolecular sensors. Enzyme activity, memory sensors, and sensors in animal models are also discussed.

We genetically encoded the photocaged amino acid 4,5-dimethoxy-2-nitrobenzylserine (DMNB-Ser, 1) in Saccharomyces cerevisiae in response to the amber nonsense codon TAG. This amino acid was converted to serine in living cells by irradiation with relatively low-energy blue light and was used to noninvasively photoactivate phosphorylation of the transcription factor Pho4, which controls the cellular response to inorganic phosphate. When substituted at phosphoserine sites that control nuclear export of Pho4, 1 blocks phosphorylation and subsequent export by the receptor Msn5 (ref. 2). We triggered phosphorylation of individual serine residues with a visible laser pulse and monitored nuclear export of Pho4-GFP fusion constructs in real time. We observed distinct export kinetics for differentially phosphorylated Pho4 mutants, which demonstrates dynamic regulation of Pho4 function. This methodology should also facilitate the analysis of other cellular processes involving free serine residues, including catalysis, biomolecular recognition and ion transport. [ABSTRACT FROM AUTHOR]

How synaptic vesicles move within central nervous synapses to their docking sites at the plasma membrane is widely discussed in synaptic physiology. This question is especially difficult to investigate in the small hippocampal boutons, which themselves can slowly move during observation in primary cell culture. Here, we describe a single particle tracking method using dual fluorescent dye labels that enabled us to visualize the movements of a single vesicle and the respective synaptic bouton simultaneously during resting conditions and stimulation. We found vesicle mobility to be very low in the absence of stimulation, in line with previous studies. Interestingly, mobility was also found to be low during synaptic activity. We found that vesicles labeled preferentially via early, late, and spontaneous endocytotic mechanisms behaved similarly at rest and during stimulation. [ABSTRACT FROM AUTHOR]

Engineering new functionality into living eukaryotic systems by enzyme evolution or de novo protein design is a formidable challenge. Cells do not rely exclusively on DNA-based evolution to generate new functionality but often utilize membrane encapsulation or formation of membraneless organelles to separate distinct molecular processes that execute complex operations. Applying this principle and the concept of two-dimensional phase separation, we develop film-like synthetic organelles that support protein translation on the surfaces of various cellular membranes. These sub-resolution synthetic films provide a path to make functionally distinct enzymes within the same cell. We use these film-like organelles to equip eukaryotic cells with dual orthogonal expanded genetic codes that enable the specific reprogramming of distinct translational machineries with single-residue precision. The ability to spatially tune the output of translation within tens of nanometers is not only important for synthetic biology but has implications for understanding the function of membrane-associated protein condensation in cells. [Display omitted] • 2D phase separation was utilized to design orthogonal enzymes • Film-like organelles maintained distinct suppressor tRNA microenvironments • Dual film-like synthetic organelles enabled orthogonal translation in eukaryotes • Cells were equipped with two expanded genetic codes in addition to the canonical one Use of 2D phase separation and two instances of codon expansion in a synthetic biology approach enables control of selective protein synthesis at the tens of nanometers scale in live cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Designer translating organelles called OTOs enable mRNA-specific protein engineering. • Different RS/tRNA pairs were tested within OTOs and found to impair OTO functionality. • Chimeric PheRS/tRNAPhe pair was optimized to restore the correct OTO performance. Site-specific incorporation of noncanonical amino acids (ncAAs) can be realized by genetic code expansion (GCE) technology. Different orthogonal tRNA synthetase/tRNA (RS/tRNA) pairs have been developed to introduce a ncAA at the desired site, delivering a wide variety of functionalities that can be installed into selected proteins. Cytoplasmic expression of RS/tRNA pairs can cause a problem with background ncAA incorporation into host proteins. The application of orthogonally translating organelles (OTOs), inspired by the concept of phase separation, provides a solution for this issue in mammalian cells, allowing site-specific and protein-selective ncAA incorporation. So far, only Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase (PylRS) has been used within OTOs, limiting the method's potential. Here, we explored the implementation of four other widely used orthogonal RS/tRNA pairs with OTOs, which, to our surprise, were unsuccessful in generating mRNA-selective GCE. Next, we tested several experimental solutions and developed a new chimeric phenylalanyl-RS/tRNA pair that enables ncAA incorporation in OTOs in a site-specific and protein-selective manner. Our work reveals unaccounted design constraints in the spatial engineering of enzyme functions using designer organelles and presents a strategy to overcome those in vivo. We then discuss current limitations and future directions of in-cell engineering in general and protein engineering using GCE specifically. [ABSTRACT FROM AUTHOR]

Over 500 natural and synthetic amino acids have been genetically encoded in the last two decades. Incorporating these noncanonical amino acids into proteins enables many powerful applications, ranging from basic research to biotechnology, materials science, and medicine. However, major challenges remain to unleash the full potential of genetic code expansion across disciplines. Here, we provide an overview of diverse genetic code expansion methodologies and systems and their final applications in prokaryotes and eukaryotes, represented by Escherichia coli and mammalian cells as the main workhorse model systems. We highlight the power of how new technologies can be first established in simple and then transferred to more complex systems. For example, whole-genome engineering provides an excellent platform in bacteria for enabling transcript-specific genetic code expansion without off-targets in the transcriptome. In contrast, the complexity of a eukaryotic cell poses challenges that require entirely new approaches, such as striving toward establishing novel base pairs or generating orthogonally translating organelles within living cells. We connect the milestones in expanding the genetic code of living cells for encoding novel chemical functionalities to the most recent scientific discoveries, from optimizing the physicochemical properties of noncanonical amino acids to the technological advancements for their in vivo incorporation. This journey offers a glimpse into the promising developments in the years to come. [ABSTRACT FROM AUTHOR]

The article presents the advantages of using the single-molecule Forster resonance energy transfer (smFRET) in analyzing the biomolecular structure and dynamics at a resolution. The process has been proven one of the most innovative approach to ensure reliability of the results compared to other micofluidic devices. It is specifically designed with enhanced photostability for improved molecular appearance.

The hallmark of eukaryotic cells is the nucleus that contains the genome, enclosed by a physical barrier known as the nuclear envelope (NE). On the one hand, this compartmentalization endows the eukaryotic cells with high regulatory complexity and flexibility. On the other hand, it poses a tremendous logistic and energetic problem of transporting millions of molecules per second across the nuclear envelope, to facilitate their biological function in all compartments of the cell. Therefore, eukaryotes have evolved a molecular "nanomachine" known as the Nuclear Pore Complex (NPC). Embedded in the nuclear envelope, NPCs control and regulate all the bi-directional transport between the cell nucleus and the cytoplasm. NPCs combine high molecular specificity of transport with high throughput and speed, and are highly robust with respect to molecular noise and structural perturbations. Remarkably, the functional mechanisms of NPC transport are highly conserved among eukaryotes, from yeast to humans, despite significant differences in the molecular components among various species. The NPC is the largest macromolecular complex in the cell. Yet, despite its significant complexity, it has become clear that its principles of operation can be largely understood based on fundamental physical concepts, as have emerged from a combination of experimental methods of molecular cell biology, biophysics, nanoscience and theoretical and computational modeling. Indeed, many aspects of NPC function can be recapitulated in artificial mimics with a drastically reduced complexity compared to biological pores. We review the current physical understanding of the NPC architecture and function, with the focus on the critical analysis of experimental studies in cells and artificial NPC mimics through the lens of theoretical and computational models. We also discuss the connections between the emerging concepts of NPC operation and other areas of biophysics and bionanotechnology. [ABSTRACT FROM AUTHOR]

The article discusses the general and efficient method for the Site-Specific Dual-Labeling of Proteins for Single Molecule Fluorescence Resonance Energy Transfer. It has showed that in vivo incorporation of a ketone amino acid provides a general strategy for site-specific labeling for proteins. It also allows proteins to be labeled in a site-specific manner because the location of a fluorophore needs to be specified by the introduction of a unique TAG codon.

Chemical biology and sensitive microscopy: How labeling and advanced microscopy techniques can synergistically drive the fi eld of biomolecular imaging. Major developments in the fi elds of protein engineering for fl uorescence labeling and advanced microscopy techniques, such as super‐resolution microscopy, or single‐molecule spectroscopy, have in the recent past allowed both areas to advance substantially. On pages 65–74, Milles and Lemke review the state of the art in both areas and evaluate how they can feedback on each other to fulfi ll the demands of precision single‐molecule studies (lower left). While the modifi cation of proteins through unnatural amino acids offers the minimal possible modifi cation to which a fl uorophore can be attached (upper left), it also has the potential of labeling within living cells. The suitability of different fl uorescent dyes for super‐resolution microscopy, as well as fl uorescence resonance energy transfer based approaches, is also reviewed with a view to multi‐parameter single molecule microscopy inside the cell (right). Cover by S. Milles and E. A. Lemke [ABSTRACT FROM AUTHOR]

Lysine acetylation is a charge‐neutralizing post‐translational modification of proteins bound by bromodomains (Brds). A 1,2,4‐triazole amino acid (ApmTri) was established as acetyllysine (Kac) mimic recruiting Brds of the BET family in contrast to glutamine commonly used for simulating this modification. Optimization of triazole substituents and side chain spacing allowed BET Brd recruitment to ApmTri‐containing peptides with affinities similar to native substrates. Crystal structures of ApmTri‐containing peptides in complex with two BET Brds revealed the binding mode which mirrored that of Kac ligands. ApmTri was genetically encoded and recombinant ApmTri‐containing proteins co‐enriched BRD3(2) from cellular lysates. This interaction was blocked by BET inhibitor JQ1. With genetically encoded ApmTri, biochemistry is now provided with a stable Kac mimic reflecting charge neutralization and Brd recruitment, allowing new investigations into BET proteins in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Die Acetylierung von Lysinen ist eine ladungsneutralisierende posttranslationale Modifikation von Proteinen, die Bromodomänen (Brds) als Bindungsmotiv dient. Eine 1,2,4‐Triazol‐Aminosäure (ApmTri) wurde als Acetyllysin (Kac)‐Mimetikum entwickelt, das Brds der BET‐Familie rekrutiert, im Gegensatz zu Glutamin, das üblicherweise zur Simulierung dieser Modifikation genutzt wird. Durch Optimierung der Triazol‐Substituenten und der Seitenkettenlänge konnten ähnliche Affinitäten von BET‐Brds zu ApmTri‐enthaltenden Peptiden wie zu nativen Substraten erreicht werden. Kristallstrukturen zweier BET‐Brds mit gebundenen ApmTri‐Peptiden zeigten einen Bindungsmechanismus, der den von gebundenen Kac‐Liganden widerspiegelte. ApmTri wurde genetisch kodiert und rekombinante ApmTri‐enthaltende Proteine konnten gemeinsam mit BRD3(2) aus Zelllysat angereichert werden. Diese Interaktion wurde durch den BET‐Inhibitor JQ1 blockiert. Die Biochemie wird durch ApmTri mit einem neuen, stabilen Kac‐Mimetikum ausgestattet, das sowohl die Ladung neutralisiert, als auch Brds rekrutiert. Dadurch werden neue Untersuchungen von BET‐Proteinen in vitro und in vivo ermöglicht. [ABSTRACT FROM AUTHOR]

In recent years, investigation of virus-cell interactions has moved from ensemble measurements to imaging analyses at the single-particle level. Advanced fluorescence microscopy techniques provide single-molecule sensitivity and subdiffraction spatial resolution, allowing observation of subviral details and individual replication events to obtain detailed quantitative information. To exploit the full potential of these techniques, virologists need to employ novel labeling strategies, taking into account specific constraints imposed by viruses, as well as unique requirements of microscopic methods. Here, we compare strengths and limitations of various labeling methods, exemplify virological questions that were successfully addressed, and discuss challenges and future potential of novel approaches in virus imaging. [ABSTRACT FROM AUTHOR]

In recent years, investigation of virus-cell interactions has moved from ensemble measurements to imaging analyses at the single-particle level. Advanced fluorescence microscopy techniques provide single-molecule sensitivity and subdiffraction spatial resolution, allowing observation of subviral details and individual replication events to obtain detailed quantitative information. To exploit the full potential of these techniques, virologists need to employ novel labeling strategies, taking into account specific constraints imposed by viruses, as well as unique requirements of microscopic methods. Here, we compare strengths and limitations of various labeling methods, exemplify virological questions that were successfully addressed, and discuss challenges and future potential of novel approaches in virus imaging. [ABSTRACT FROM AUTHOR]

Ataxin-2, an RNA-binding protein that is conserved across eukaryotes, is involved in stress granule assembly and age-associated neurodegenerative diseases. In this issue of Molecular Cell , Boeynaems et al. 1 identify a short linear motif in ataxin-2 as a condensation switch, providing molecular insights into its essential role in cellular stress response. Ataxin-2, an RNA-binding protein that is conserved across eukaryotes, is involved in stress granule assembly and age-associated neurodegenerative diseases. In this issue of Molecular Cell , Boeynaems et al.1 identify a short linear motif in ataxin-2 as a condensation switch, providing molecular insights into its essential role in cellular stress response. [ABSTRACT FROM AUTHOR]

Abstract: Optical nanoscopy techniques using localization based image reconstruction, also termed super-resolution microscopy (SRM), have become a standard tool to bypass the diffraction limit in fluorescence light microscopy. The localization precision measured for the detected fluorophores is commonly used to describe the maximal attainable resolution. However, this measure takes not all experimental factors, which impact onto the finally achieved resolution, into account. Several other methods to measure the resolution of super-resolved images were previously suggested, typically relying on intrinsic standards, such as molecular rulers, or on a priori knowledge about the specimen, e.g. its spatial frequency content. Here we show that Fourier ring correlation provides an easy-to-use, laboratory consistent standard for measuring the resolution of SRM images. We provide a freely available software tool that combines resolution measurement with image reconstruction. [Copyright &y& Elsevier]

Biological activity in proteins requires them to share the energy landscape for folding and global conformational motions. 2 key determinants of function. Although most structural studies to date have focused on fluctuations around a single structural basin, we directly observe the coexistence of 2 symmetrically opposed conformations for a mutant of the Rop-homodimer (Repressor of Primer) in single-molecule fluorescence resonance energy transfer (5mFRET) measurements. We find that mild denaturing conditions can affect the sensitive balance between the conformations, generating an equilibrium ensemble consisting of 2 equally occupied structural basins. Despite the need for large-scale conformational rearrangement, both native structures are dynamically and reversibly adopted for the same paired molecules without separation of the constituent monomers. Such an ability of some proteins or protein complexes to switch between conformations by thermal fluctuations and/or minor environmental changes could be central to their ability to control biological function. [ABSTRACT FROM AUTHOR]

We studied the coupled binding and folding of α-synuclein. an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and dynamics. Results revealed an intricate energy landscape on which binding of α-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple α-helical structures. α-synuclein conformation is not continuously tunable, but instead partitions into 2 main classes of folding landscape structural minima. The switch between a broken and an extended helical structure can be triggered by changing the concentration of binding partners or by varying the curvature of the binding surfaces presented by micelles or bilayers composed of the lipid-mimetic SDS. Single-molecule experiments with lipid vesicles of various composition showed that a low fraction of negatively charged lipids, similar to that found in biological membranes, was sufficient to drive α-synuclein binding and folding, resulting here in the induction of an extended helical structure. Overall, our results imply that the 2 folded structures are preen- coded by the α-synuclein amino acid sequence, and are tunable by small-molecule supramolecular states and differing membrane properties, suggesting novel control elements for biological and amyloid regulation of α-synuclein. [ABSTRACT FROM AUTHOR]

The yeast prion protein Sup35 is a translation termination factor. whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to "burst-phase" intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was ≈43 Å. This increased with denaturant in a noncooperative fashion to ≈63 Å, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. Fluorescence correlation spectroscopy analysis of singly labeled NM revealed fast conformational fluctuations on the 20- to 300-ns time scale. Quenching from proximal and distal tyrosines resulted in distinct fast and slower fluctuations. Our results indicate that native monomeric NM is composed of an ensemble of structures, having a collapsed and rapidly fluctuating N region juxtaposed with a more extended M region. The stability of such ensembles is likely to play a key role in prion conversion. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells. [Display omitted] • We constructed an intact amber-free HIV-1 provirus for in-virus click labeling • System enables dual-ncAA insertion for biorthogonal click labeling on intact virions • smFRET of Env reinforced its structural dynamics observed via conventional labeling • Live cell imaging tracked amber-free click-labeled Env virions in cells Ao et al. developed an intact amber-free HIV-1 system that enables minimally invasive Env tagging by genetic code expansion and in-virus bioorthogonal click labeling for single-molecule FRET structural dynamics and advanced microscopy studies of virus entry, trafficking, and egress in living cells. [ABSTRACT FROM AUTHOR]

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 β‐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 β‐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. [ABSTRACT FROM AUTHOR]

Nucleoporins (Nups) represent a range of proteins most known for composing the macromolecular assembly of the nuclear pore complex (NPC). Among them, the family of intrinsically disordered proteins (IDPs) phenylalanine-glycine (FG) rich Nups, form the permeability barrier and coordinate the high-speed nucleocytoplasmic transport in a selective way. Those FG-Nups have been demonstrated to participate in various biological processes besides nucleocytoplasmic transport. The high number of accessible hydrophobic motifs of FG-Nups potentially gives rise to this multifunctionality, enabling them to form unique microenvironments. In this review, we discuss the multifunctionality of disordered and F-rich Nups and the diversity of their localizations, emphasizing the important roles of those Nups in various regulatory and metabolic processes. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the site-specific genetic incorporation of an environmentally sensitive, fluorescent amino acid into proteins in Saccharomyces cerevisiae, an orthogonal Escherichia coli amber suppressor tRNA. The study utilized a method that serves as a local probe of conformational changes in the structure of protein caused by ligand binding. The study has also incorporated fluorescent amino acid into proteins in yeast.

Nuclear pore complexes (NPCs) regulate all cargo traffic across the nuclear envelope. The transport conduit of NPCs is highly enriched in disordered phenylalanine/glycine-rich nucleoporins (FG-Nups), which form a permeability barrier of still elusive and highly debated molecular structure. Here we present a microfluidic device that triggered liquid-to-liquid phase separation of FG-Nups, which yielded droplets that showed typical properties of a liquid state. On the microfluidic chip, droplets were perfused with different transport-competent or -incompetent cargo complexes, and then the permeability barrier properties of the droplets were optically interrogated. We show that the liquid state mimics permeability barrier properties of the physiological nuclear transport pathway in intact NPCs in cells: that is, inert cargoes ranging from small proteins to large capsids were excluded from liquid FG-Nup droplets, but functional import complexes underwent facilitated import into droplets. Collectively, these data provide an experimental model of how NPCs can facilitate fast passage of cargoes across an order of magnitude in cargo size. [ABSTRACT FROM AUTHOR]

A summary is presented of an article published online at http://dx.doi.org/10.1126/science.aaw2644 on the creation of organelles which can enable reassignment of codons of selected messenger RNAs in eukaryotes. It states that selective and specific protein translation can be created within orthogonally translating organelles. It comments that cells with the organelle were able to selectively and efficiently perform Amber suppression of targeted messenger RNA.

In diesem Beitrag wird das Zusammenspiel von Dynamik und Molekülerkennung bei intrinsisch ungeordneten Proteinen (IDPs) durch die Anwendung von zeitauflösenden Messtechniken im fs‐ bis ns‐Bereich beleuchtet. Die Konformations‐ und Wasserdynamik an Grenzflächen sowie deren Abschwächung durch die Bindung an ein Partnermolekül wurde anhand zweier IDPs, IBB und Nup153FG, untersucht. Beide Proteine binden an denselben Transportrezeptor (Importinβ), jedoch durch grundlegend verschiedene Bindungsmechanismen. Die Fluktuation des Lösungsmittels an der dynamischen Grenzfläche des Nup153FG‐Importinβ‐Komplexes blieb weitgehend ungestört und beschleunigte sich leicht im Vergleich zum ungebundenen Zustand. Im Gegensatz dazu wurde im IBB‐Importinβ‐Komplex eine beträchtliche relative Verlangsamung der Wasserdynamik an der starreren Grenzfläche beobachtet. Die Ergebnisse zeigen eine Korrelation zwischen der Hydratationsdynamik an Grenzflächen und der Plastizität von IDP‐Komplexen, was eine funktionelle Relevanz einer solchen Regulation impliziert, insbesondere auch für den nukleozytoplasmatischen Transport. Der Tanz des Wassers: Die Dynamik von Wasser an Grenzflächen von Komplexen mit intrinsisch ungeordneten Proteinen wurde mit einer Kombination spektroskopischer Methoden auf der Femto‐ bis Nanosekunden‐Zeitskala untersucht. Die Ergebnisse liefern neue Einsichten, wie sich die Solvatationsdynamik in Abhängigkeit vom Bindungsmechanismus verändert. Dabei liegt der Fokus auf Proteinen, die eine wichtige Rolle im Kerntransport spielen. [ABSTRACT FROM AUTHOR]

The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time‐resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinβ) with drastically different binding mechanisms, coupled folding–binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG‐Importinβ fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB‐Importinβ complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport. The dance of water: Water dynamics at the interface of intrinsically disordered proteins was studied on the femto‐ to nanosecond timescale by using a combination of advanced site‐specific spectroscopic tools. The results offer new insight into the relationship between solvation dynamics and the binding mechanism of proteins of central relevance to nuclear transport. [ABSTRACT FROM AUTHOR]

Chemically produced, accurately linkable oligosaccharides are of importance for the synthesis of neo‐glycoproteins. On the route to high‐mannose type N‐glycans, we present a convenient synthesis of several glycans bearing an azide moiety at the reducing end. An azido‐glycan core structure as valuable precursor was modified into the protected N‐glycan pentasaccharide core structure and the possibility of modular attachment of different antenna was demonstrated through synthesis of a pentamannose donor and glycosylation with the core structure. The azido function allows for chemical ligation with recombinantly modified proteins featuring noncanonical cyclooctyne amino acids, providing access to customized glycopatterns of glycoproteins, e.g., of antibodies that are of high interest for biopharmaceutical applications. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Protein bioconjugation frequently makes use of aldehydes as reactive handles, with methods for their installation being highly valued. Here a new, powerful strategy to unmask a reactive protein aldehyde is presented. A genetically encoded caged glyoxyl aldehyde, situated in solvent-accessible locations, can be rapidly decaged through treatment with just one equivalent of allylpalladium(ii) chloride dimer at physiological pH. The protein aldehyde can undergo subsequent oxime ligation for site-selective protein modification. Quick yet mild conditions, orthogonality and powerful exposed reactivity make this strategy of great potential in protein modification. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Huntington's disease is caused by expansion of a polyglutamine (polyQ) domain within exon 1 of the huntingtin gene (Httex1). The prevailing hypothesis is that the monomeric Httex1 protein undergoes sharp conformational changes as the polyQ length exceeds a threshold of 36-37 residues. Here, we test this hypothesis by combining novel semi-synthesis strategies with state-of-the-art single-molecule Foörster resonance energy transfer measurements on biologically relevant, monomeric Httex1 proteins of five different polyQ lengths. Our results, integrated with atomistic simulations, negate the hypothesis of a sharp, polyQ length-dependent change in the structure of monomeric Httex1. Instead, they support a continuous global compaction with increasing polyQ length that derives from increased prominence of the globular polyQ domain. Importantly, we show that monomeric Httex1 adopts tadpole-like architectures for polyQ lengths below and above the pathological threshold. Our results suggest that higher order homotypic and/or heterotypic interactions within distinct sub-populations of neurons, which are inevitable at finite cellular concentrations, are likely to be the main source of sharp polyQ length dependencies of HD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Site-directed spin labeling for EPR- and NMR spectroscopy has mainly been achieved exploiting the specific reactivity of cysteines. For proteins with native cysteines or for in vivo applications, an alternative coupling strategy is required. In these cases click chemistry offers major benefits by providing a fast and highly selective, biocompatible reaction between azide and alkyne groups. Here, we establish click chemistry as a tool to target unnatural amino acids in vitro and in vivo using azide- and alkyne-functionalized spin labels. The approach is compatible with a variety of labels including reduction-sensitive nitroxides. Comparing spin labeling efficiencies from the copper-free with the strongly reducing copper(I)-catalyzed azide-alkyne click reaction, we find that the faster kinetics for the catalyzed reaction outrun reduction of the labile nitroxide spin labels and allow quantitative labeling yields within short reaction times. Inter-spin distance measurements demonstrate that the novel side chain is suitable for paramagnetic NMR- or EPR-based conformational studies of macromolecular complexes. [ABSTRACT FROM AUTHOR]