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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]

We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein ?-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid mimics and subsequent rapid formation of transient structures in the encounter complex. The method also enabled analysis of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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]

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]

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]

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]

Editors at Science requested our input on the above discussion (comment by Best et al. and response by Riback et al.) because both sets of authors use our data from Fuertes et al. (2017) to support their arguments. The topic of discussion pertains to the discrepant inferences drawn from SAXS versus FRETmeasurements regarding the dimensions of intrinsically disordered proteins (IDPs) in aqueous solvents. Using SAXS measurements on labeled and unlabeled proteins, we ruled out the labels used for FRETmeasurements as the cause of discrepant inferences between the two methods. Instead, we propose that FRET and SAXS provide complementary readouts because of a decoupling of size and shape fluctuations that is intrinsic to finite-sized, heteropolymeric IDPs. Accounting for this decoupling resolves the discrepant inferences between the two methods, thus making a case for the utility of both methods. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block-although compositionally identical-engage in different local sets of interactions and conformations. [ABSTRACT FROM AUTHOR]

Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-determining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aß, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Aß, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike. [ABSTRACT FROM AUTHOR]

Summary Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static “spring-loaded” fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of pH and receptor binding shifts the dynamic equilibrium of HA2 in favor of forward progression along the membrane-fusion reaction coordinate. Interaction with the target membrane promotes irreversible transition of HA2 to the post-fusion state. The reversibility of HA2 conformation may protect against transition to the post-fusion state prior to arrival at the target membrane. [ABSTRACT FROM AUTHOR]

Summary The envelope glycoproteins (Env) of HIV-1 mediate cell entry through fusion of the viral envelope with a target cell membrane. Intramembrane mobility and clustering of Env trimers at the viral budding site are essential for its function. Previous live-cell and super-resolution microscopy studies were limited by lack of a functional fluorescent Env derivative, requiring antibody labeling for detection. Introduction of a bio-orthogonal amino acid by genetic code expansion, combined with click chemistry, offers novel possibilities for site-specific, minimally invasive labeling. Using this approach, we established efficient incorporation of non-canonical amino acids within HIV-1 Env in mammalian cells. The engineered protein retained plasma membrane localization, glycosylation, virion incorporation, and fusogenic activity, and could be rapidly and specifically labeled with synthetic dyes. This strategy allowed us to revisit Env dynamics and nanoscale distribution at the plasma membrane close to its native state, applying fluorescence recovery after photo bleaching and STED nanoscopy, respectively. [ABSTRACT FROM AUTHOR]

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 challenges, there is critical need for further improvement. 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: “E.A.L. holds patents related to OTOs. M.E.S. and M.J. declare no competing interests.”., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s).)

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol 1 . The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs) 2,3 . Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs-about 50 MDa-is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence 4-11 . Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides-in the terminology of the Flory polymer theory 12 -a 'good solvent' environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder-function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry., (© 2023. The Author(s).)

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology., (© 2023. The Author(s).)

Engineering new functionalities into living eukaryotic systems is one of the main goals of synthetic biology. To this end, often enzyme evolution or de novo protein design is employed, which each have their own advantages and disadvantages. As complimentary tools, we recently developed orthogonally translating and film-like synthetic organelles that allow to create new enzyme functionalities based on spatial separation. We applied this technology to genetic code expansion (GCE) and showed that it is possible to equip eukaryotic cells with multiple orthogonal genetic codes that enable the specific reprogramming of distinct translational machineries, each with single-residue precision.In this protocol, we describe how synthetic organelles can be used to perform mRNA selective GCE and how they can be further developed to allow the simultaneous incorporation of distinct noncanonical amino acids (ncAAs) into selected proteins and how this can be used to label proteins selectively with fluorescent dyes via bioorthogonal chemistry., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Condensate formation of biopolymer solutions, prominently those of various intrinsically disordered proteins (IDPs), is often driven by "sticky" interactions between associating residues, multivalently present along the polymer backbone. Using a ternary mean-field "stickers-and-spacers" model, we demonstrate that if sticker association is of the order of a few times the thermal energy, a delicate balance between specific binding and nonspecific polymer-solvent interactions gives rise to a particularly rich ternary phase behavior under physiological circumstances. For a generic system represented by a solution comprising multiassociative scaffold and client polymers, the difference in solvent compatibility between the polymers modulates the nature of isothermal liquid-liquid phase separation (LLPS) between associative and segregative. The calculations reveal regimes of dualistic phase behavior, where both types of LLPS occur within the same phase diagram, either associated with the presence of multiple miscibility gaps or a flip in the slope of the tie-lines belonging to a single coexistence region.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: C.D.R. and E.A.L. have filed a patent application on OT organelle technology (EP 19157257.7)., (Copyright © 2021 Elsevier Ltd. All rights reserved.)