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Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic and diagnostic applications. Here, we use genetic code expansion to site-specifically modify target proteins with an isopeptide-linked glycylglycine moiety that serves as an acceptor nucleophile in AEPmediated transpeptidation with various probes containing a tripeptidic recognition motif. Our approach allows simple and flexible labeling of recombinant proteins at any internal site and leaves a minimal, entirely peptidic footprint (NGG) in the conjugation product. We show site-specific labeling of diverse target proteins with various biophysical probes, including dual labeling at an internal site and the N-terminus. Furthermore, we harness AEP-mediated transpeptidation for generation of ubiquitinand ubiquitin-like-modifier conjugates bearing a native isopeptide bond and only one point mutation in the linker region., Journal of the American Chemical Society, 144 (29), ISSN:0002-7863, ISSN:1520-5126

Approaches for profiling protease substrates are critical for defining protease functions, but remain challenging tasks. We combine genetic code expansion, photocrosslinking and proteomics to identify substrates of the mitochondrial (mt) human caseinolytic protease P (hClpP). Site-specific incorporation of the diazirine-bearing amino acid DiazK into the inner proteolytic chamber of hClpP, followed by UV-irradiation of cells, allows to covalently trap substrate proteins of hClpP and to substantiate hClpP's major involvement in maintaining overall mt homeostasis. In addition to confirming many of the previously annotated hClpP substrates, our approach adds a diverse set of new proteins to the hClpP interactome. Importantly, our workflow allows identifying substrate dynamics upon application of external cues in an unbiased manner. Identification of unique hClpP-substrate proteins upon induction of mt oxidative stress, suggests that hClpP counteracts oxidative stress by processing of proteins that are involved in respiratory chain complex synthesis and maturation as well as in catabolic pathways., Angewandte Chemie. International Edition, 61 (10), ISSN:1433-7851, ISSN:1521-3773, ISSN:0570-0833

Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and β subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate β subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone–client complexes for mass spectrometry. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:β assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones., Journal of Biological Chemistry, 298 (12), ISSN:0021-9258, ISSN:1083-351X

Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals., Light‐induced protein labeling: Cyclopropenone‐caged dibenzoannulated bicyclononynes (photo‐DMBO) are photo‐activatable dienophiles that engage in rapid inverse electron‐demand Diels–Alder cycloadditions with tetrazines upon light‐induced decarbonylation. Site‐specific incorporation of methyl‐tetrazine amino acids allows photo‐induced protein labeling in living cells with spatio‐temporal control using photo‐DMBO fluorophore conjugates.

Post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubls) is central to the regulation of eukaryotic cellular processes. Our ability to study the effects of ubiquitylation, however, is limited by the difficulty to prepare homogenously modified proteins in vitro and by the impossibility to selectively trigger specific ubiquitylation events in living cells. Here we combine genetic-code expansion, bioorthogonal Staudinger reduction and sortase-mediated transpeptidation to develop a general tool to ubiquitylate proteins in an inducible fashion. The generated ubiquitin conjugates display a native isopeptide bond and bear two point mutations in the ubiquitin C terminus that confer resistance toward deubiquitinases. Nevertheless, physiological integrity of sortase-generated diubiquitins in decoding cellular functions via recognition by ubiquitin-binding domains is retained. Our approach allows the site-specific attachment of Ubls to nonrefoldable, multidomain proteins and enables inducible and ubiquitin-ligase-independent ubiquitylation of proteins in mammalian cells, providing a powerful tool to dissect the biological functions of ubiquitylation with temporal control.

Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome‑wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome‑wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the N‑terminus proteome‑wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.

The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates, Nucleic Acids Research, 49 (11), ISSN:1362-4962, ISSN:0301-5610

Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups. Rooted in the principles of physical organic chemistry, bioorthogonal reactions are intrinsically selective transformations not commonly found in biology. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide–alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions. Bioorthogonal chemistry has significant overlap with the broader field of ‘click chemistry’ — high-yielding reactions that are wide in scope and simple to perform, as recently exemplified by sulfuryl fluoride exchange chemistry. The underlying mechanisms of these transformations and their optimal conditions are described in this Primer, followed by discussion of how bioorthogonal chemistry has become essential to the fields of biomedical imaging, medicinal chemistry, protein synthesis, polymer science, materials science and surface science. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody–drug conjugation and drug delivery. This Primer describes standards for reproducibility and data deposition, outlines how current limitations are driving new research directions and discusses new opportunities for applying bioorthogonal chemistry to emerging problems in biology and biomedicine.

Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome‑wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome‑wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the N‑terminus proteome‑wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.

Labelling proteins at internal sites holds promise for generating novel protein conjugates in a programmable fashion. Now, a chemoenzymatic approach, dubbed LACE, enables the site-specific modification of recombinant proteins that contain a short genetically encoded tag.

In recent years it has become possible to genetically encode an expanded set of designer amino acids with tailored chemical and physical properties (dubbed unnatural amino acids, UAAs) into proteins in living cells by expanding the genetic code. Together with developments in chemistries that are amenable to and selective within physiological settings, these strategies have started to have a big impact on biological studies, as they enable exciting in cellulo applications. Here we highlight recent advances to covalently stabilize transient protein-protein interactions and capture enzyme substrate-complexes in living cells using proximity-triggered and residue-selective photo-induced crosslinking approaches. Furthermore, we describe recent efforts in controlling enzyme activity with photocaged UAAs and in extending their application to a variety of enzymatic scaffolds. In addition, we discuss the site-specific incorporation of UAAs mimicking post-translational modifications (PTMs) and approaches to generate natively-linked ubiquitin-protein conjugates to probe the role of PTMs in modulating complex cellular networks.

Our understanding of sequence variation in the HLA-DPB1 gene is largely restricted to the hypervariable antigen recognition domain (ARD) encoded by exon 2. Here, we employed a redundant sequencing strategy combining long-read and short-read data to accurately phase and characterise in full length the majority of common and well-documented (CWD) DPB1 alleles as well as alleles with an observed frequency of at least 0.0006% in our predominantly European sample set. We generated 664 DPB1 sequences, comprising 279 distinct allelic variants. This allows us to present the, to date, most comprehensive analysis of the nature and extent of DPB1 sequence variation. The full-length sequence analysis revealed the existence of two highly diverged allele clades. These clades correlate with the rs9277534 A → G variant, a known expression marker located in the 3 ′ -UTR. The two clades are fully differentiated by 174 fixed polymorphisms throughout a 3.6 kb stretch at the 3 ′ -end of DPB1. The region upstream of this differentiation zone is characterised by increasingly shared variation between the clades. The low-expression A clade comprises 59% of the distinct allelic sequences including the three by far most frequent DPB1 alleles, DPB1*04:01, DPB1*02:01 and DPB1*04:02. Alleles in the A clade show reduced nucleotide diversity with an excess of rare variants when compared to the high-expression G clade. This pattern is consistent with a scenario of recent proliferation of A-clade alleles. The full-length characterisation of all but the most rare DPB1 alleles will benefit the application of NGS for DPB1 genotyping and provides a helpful framework for a deeper understanding of high- and low-expression alleles and their implications in the context of unrelated haematopoietic stem-cell transplantation.

We report the genetically encoded incorporation of phenylacetyl protected lysine (PacK) into proteins in Escherichia coli. This unnatural side-chain modification can be enzymatically removed using either penicillin G acylase (PGA) or, surprisingly, the sirtuin SrtN from Bacillus subtilis. Our approach expands the toolbox to reversibly control protein structure and function under very mild and non-denaturing conditions, as demonstrated by triggering the activity of the nonribosomal peptide synthetase GrsA.

Methylation of a conserved lysine in C-terminal domain of the molecular chaperone Hsp90 was shown previously to affect its in vivo function. However, the underlying mechanism remained elusive. Through a combined experimental and computational approach, this study shows that this site is very sensitive to sidechain modifications and crucial for Hsp90 activity in vitro and in vivo. Our results demonstrate that this particular lysine serves as a switch point for the regulation of Hsp90 functions by influencing its conformational cycle, ATPase activity, co-chaperone regulation, and client activation of yeast and human Hsp90. Incorporation of the methylated lysine via genetic code expansion specifically shows that upon modification, the conformational cycle of Hsp90 is altered. Molecular dynamics simulations including the methylated lysine suggest specific conformational changes that are propagated through Hsp90. Thus, methylation of the C-terminal lysine allows a precise allosteric tuning of Hsp90 activity via long distances., Methylation of a lysine residue in Hsp90 is a recently discovered post-translational modification but the mechanistic effects of this modification have remained unknown so far. Here the authors combine biochemical and biophysical approaches, molecular dynamics (MD) simulations and functional experiments with yeast and show that this lysine is a switch point, which specifically modulates conserved Hsp90 functions including co-chaperone regulation and client activation.

Protein-protein interactions are central to many biological processes. A considerable challenge consists however in understanding and deciphering when and how proteins interact, and this can be particularly difficult when interactions are weak and transient. The site-specific incorporation of unnatural amino acids (UAAs) that crosslink with nearby molecules in response to light provides a powerful tool for mapping transient protein-protein interactions and for defining the structure and topology of protein complexes both in vitro and in vivo. Complementary strategies consist in site-specific incorporation of UAAs bearing electrophilic moieties that react with natural nucleophilic amino acids in a proximity-dependent manner, thereby chemically stabilizing low-affinity interactions and providing additional constraints on distances and geometries in protein complexes. Herein, we review how UAAs bearing fine-tuned chemical moieties that react with proteins in their vicinity can be utilized to map, study, and characterize weak and transient protein-protein interactions in living systems.

The risk of acute graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation is increased with donor-recipient HLA-DPB1 allele mismatching. The single-nucleotide polymorphism (SNP) rs9277534 within the 3′ untranslated region (UTR) correlates with HLA-DPB1 allotype expression and serves as a marker for permissive HLA-DPB1 mismatches. Since rs9277534 is not routinely typed, we analyzed 32,681 samples of mostly European ancestry to investigate if the rs9277534 allele can be reliably imputed from standard DPB1 genotyping. We confirmed the previously-defined linkages between rs9277534 and 18 DPB1 alleles and established additional linkages for 46 DPB1 alleles. Based on these linkages, the rs9277534 allele could be predicted for 99.6% of the samples based on DPB1 genotypes (99.99% concordance). We demonstrate that 100% prediction accuracy could be achieved if the prediction utilized exon 3 sequence information. DPB1 genotyping based on exon 2 data alone allows no unambiguous rs9277534 allele prediction but was estimated to maintain 99% accuracy for samples of European descent. We conclude that DPB1 genotyping is sufficient to infer the DPB1 expression marker rs9277534 with high accuracy. This information could be used to select donors with permissive HLA-DPB1 mismatches without directly screening for rs9277534.

The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions in vitro and in vivo.

Fluoreszierende Proteine und neue Fluoreszenzfarbstoffe machen Proteine in lebenden Zellen fur neue Mikroskopietechniken sichtbar. Die Optogenetik setzt kunstliche Photorezeptoren ein, um Ablaufe in Zellen mit Licht zu steuern. Mehr als 30 Patente zu Crispr-Cas-Anwendungen sind mittlerweile eingereicht, die ersten klinischen Studien mit Cas9-modifizierten humanen Zellen laufen. Einige Krankheiten, darunter Brustkrebs und Tuberkulose, hangen mit RNA-Modifikationen zusammen. Modifizerte RNA-Nukleoside sind mit HPLC-MS detektierbar. Bei vielen Erkrankungen spielen posttranslationale Modifikationen an Proteinen eine zentrale Rolle. Nichtribosomale Peptidsynthetasen konnen riesig sein und setzen ganz unterschiedliche Bausteine zusammen.

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio‐orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence‐based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse‐chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis., ChemBioChem, 21 (13), ISSN:1439-4227, ISSN:1439-7633

The biotin-streptavidin interaction is among the strongest known in nature. Herein, the site-directed incorporation of biotin and 2-iminobiotin composed of noncanonical amino acids (ncAAs) into proteins is reported. 2-Iminobiotin lysine was employed for protein purification based on the pH-dependent dissociation constant to streptavidin. By using the high-affinity binding of biotin lysine, the bacterial protein RecA could be specifically isolated and its interaction partners analyzed. Furthermore, the biotinylation approach was successfully transferred to mammalian cells. Stringent control over the biotinylation site and the tunable affinity between ncAAs and streptavidin of the different biotin analogues make this approach an attractive tool for protein interaction studies, protein immobilization, and the generation of well-defined protein-drug conjugates.

The past 10 years have seen a significant and rapid development in chemoselective reactions for labeling and controlling biological molecules in vitro but also in vivo. Here we review the class of inverse-electron-demand Diels–Alder (iEDDA) cycloadditions that have been re-discovered and developed in recent years as ideal bioorthogonal reactions and that by now play an important role in modern bioconjugation chemistry. We will especially focus on iEDDA reactions between 1,2,4,5-tetrazines and strained dienophiles and we will describe how they can be used for imaging, manipulating, and detecting biological processes. 1 Introduction 2 Inverse-Electron-Demand Diels–Alder (iEDDA) Cycloadditions 3 Synthesis of 1,2,4,5-Tetrazines 4 Synthesis of Strained Dienophiles 5 Applications in Biology 5.1 iEDDA Cycloadditions on Proteins 5.1.1 Fluorescence Labeling of Proteins via Genetic Approaches 5.1.2 Controlling Enzyme Activity in Live Cells via iEDDA Reactions 5.1.3 In vivo Imaging of Proteins via Modified Antibodies and Small Molecules 5.2 iEDDA Reactions on Nucleic Acids 5.3 iEDDA Cycloadditions To Label Glycans and Lipids 5.4 Orthogonal Bioorthogonal Reactions 5.5 Tetrazine Decaging Reactions 6 Recent Development of Other iEDDA Bioorthogonal Reactions 7 Conclusions and Outlook

Bilateral Symposium: The Shanghai Institute of Organic Chemistry (SIOC) hosted the 6th Sino-German Frontiers of Chemistry Symposium from August 29 to September 3, 2016. This event, co-organized by the Chinese Chemical Society (CCS) and the Gesellschaft Deutscher Chemiker (German Chemical Society), featured excellent talks by the invited early-career chemists of both countries and offered ample opportunities for discussions and making friends among the participants.

The killer-cell immunoglobulin-like receptor (KIR) genes regulate natural killer cell activity, influencing predisposition to immune mediated disease, and affecting hematopoietic stem cell transplantation (HSCT) outcome. Owing to the complexity of the KIR locus, with extensive gene copy number variation (CNV) and allelic diversity, high-resolution characterization of KIR has so far been applied only to relatively small cohorts. Here, we present a comprehensive high-throughput KIR genotyping approach based on next generation sequencing. Through PCR amplification of specific exons, our approach delivers both copy numbers of the individual genes and allelic information for every KIR gene. Ten-fold replicate analysis of a set of 190 samples revealed a precision of 99.9%. Genotyping of an independent set of 360 samples resulted in an accuracy of more than 99% taking into account consistent copy number prediction. We applied the workflow to genotype 1.8 million stem cell donor registry samples. We report on the observed KIR allele diversity and relative abundance of alleles based on a subset of more than 300,000 samples. Furthermore, we identified more than 2,000 previously unreported KIR variants repeatedly in independent samples, underscoring the large diversity of the KIR region that awaits discovery. This cost-efficient high-resolution KIR genotyping approach is now applied to samples of volunteers registering as potential donors for HSCT. This will facilitate the utilization of KIR as additional selection criterion to improve unrelated donor stem cell transplantation outcome. In addition, the approach may serve studies requiring high-resolution KIR genotyping, like population genetics and disease association studies.

Oxford Nanopore Technologies’ MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunological compatibility between organ donors and recipients, but the platform’s high error rate makes it challenging to type alleles with accuracy. Here, we developed and validated accurate typing of HLA by Oxford nanopore (Athlon), a bioinformatic pipeline that i) maps nanopore reads to a database of known HLA alleles, ii) identifies candidate alleles with the highest read coverage at different resolution levels that are represented as branching nodes and leaves of a tree structure, iii) generates consensus sequences by remapping the reads to the candidate alleles, and iv) calls the final diploid genotype by blasting consensus sequences against the reference database. Using two independent datasets generated on the R9.4 flow cell chemistry, Athlon achieved a 100% accuracy in class I HLA typing at the 2-field resolution.

Nanopore sequencing, a paradigm change in sequencing technologies, offers a new cost-effective and scalable platform for HLA genotyping. Among the new generation of high-throughput sequencing technologies, the MinION nanopore sequencer is the first to offer a non-template-based direct DNA sensing sequencing technology. Oxford Nanopore Technologies (ONT) introduced the first version of the MinION in 2014; since then, the platform has gone through multiple iterations resulting in higher throughput and sequencing accuracy. The "what you put in is what you get" nature of the platform enables molecules to be sequenced without fragmentation. This results in ultra-long read lengths in the order of tens of kilobases enabling entire genes to be characterized with fully phased sequence information. With release R9.5, the MinION platform has reached a quality that enables HLA genotyping with minor shortcomings in long homopolymer regions. Within this chapter, we describe a protocol for sequencing and genotyping HLA Class I alleles using the MinION.

Graphical Abstract, Efficient access to proteins modified site-specifically with glycans is important in glycobiology and for therapeutic applications. Herein, we report a biocompatible protein glycoconjugation by inverse demand Diels–Alder reaction between tetrazine and trans-cyclooctene. Tetrazine functionalized glycans were obtained in one step by CuAAC (Cu-catalyzed alkyne azide cycloaddition) between glycosyl azide and an alkyne-tetrazine adduct. Site-specific glycoconjugation was performed chemoselectively on a target protein in which a trans-cyclooctene derivatized lysine was genetically encoded. Glycoconjugation proceeded to completion on purified protein and was shown to be selective for the target protein in E. coli.

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate th...

Two cores were drilled on rock glacier Lazaun in the southern Ötztal Alps (N Italy). The average ice content of core Lazaun I is 43 vol.% and of core Lazaun II is 22 vol.%. Radiocarbon dating of plant macrofossil remains of core Lazaun I yielded ages ranging from 8960 cal yr BP at a depth of ca. 23.5 m to 2240 cal yr BP at a depth of 2.8 m, indicating that the ice near the base is approximately 10,300 yr old. The rock glacier was intact since that time and the ice persisted even during warm periods of the Holocene. An ice-free debris layer between 16.8 and 14.7 m separates the rock glacier into two frozen bodies. Inclinometer measurements indicate that both frozen bodies are active and that deformation occurs within a shear horizon at a depth of 20–25 m at the base of the lower frozen body and to a minor extent at a depth of approximately 14 m at the base of the upper frozen body. The ice-free debris layer in the middle of the Lazaun rock glacier indicates a more than five centennial long drought period, which dates to about 4300–3740 cal yr BP.

Nanopore sequencing, a paradigm change in sequencing technologies, offers a new cost-effective and scalable platform for HLA genotyping. Among the new generation of high-throughput sequencing technologies, the MinION nanopore sequencer is the first to offer a non-template-based direct DNA sensing sequencing technology. Oxford Nanopore Technologies (ONT) introduced the first version of the MinION in 2014; since then, the platform has gone through multiple iterations resulting in higher throughput and sequencing accuracy. The "what you put in is what you get" nature of the platform enables molecules to be sequenced without fragmentation. This results in ultra-long read lengths in the order of tens of kilobases enabling entire genes to be characterized with fully phased sequence information. With release R9.5, the MinION platform has reached a quality that enables HLA genotyping with minor shortcomings in long homopolymer regions. Within this chapter, we describe a protocol for sequencing and genotyping HLA Class I alleles using the MinION.

The desire to study and manipulate biological processes in their native environment has fueled the development of approaches to endow proteins with new chemical groups in vivo. Genetic code expansion allows the site-specific incorporation of artificial, designer amino acids into virtually any protein in living cells and animals. Together with significant developments in designing and rediscovering chemistries that are amenable to physiological conditions and applicable in living systems (in vivo chemistries), these methods have begun to have a direct impact on studies of biological processes that are difficult or impossible to address by more traditional approaches. An emerging area with enormous potential is the site-specific incorporation of unnatural amino acids with functional groups that allow subsequent covalent modification of the protein with biophysical probes and small molecules via chemoselective reactions. This provides an important approach for imaging protein location in live cells and can also be used for controlling an enzyme's activity in vivo. Likewise, the co-translational site-specific incorporation of unnatural amino acids bearing new functional groups that are inert under physiological conditions, but become reactive to form covalent linkages with biomolecules in their vicinity upon a certain trigger, has become an important approach for understanding protein–protein interactions in live cells. This chapter discusses how genetic code expansion approaches together with in vivo chemistries can be utilized to introduce artificial covalent linkages into proteins and how these methods can be used for studying and controlling protein localization and activity and also for mapping and characterizing protein–protein interactions.

Oxford Nanopore Technologies’ MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunological compatibility between organ donors and recipients, but the platform’s high error rate makes it challenging to type alleles with clinical-grade accuracy. Here, we developed and validated Athlon, an algorithm that iteratively scores nanopore reads mapped to a hierarchical database of HLA alleles to arrive at a consensus diploid genotype; Athlon achieved a 100% accuracy in class I HLA typing at high resolution.

Homozygous carriers of CCR5-Δ32, a gene variant of CC-type chemokine receptor 5 (CCR5), are highly resistant to infections with human immunodeficiency virus type 1 (HIV-1) and therefore preferred stem cell donors for HIV-infected patients. We analyzed CCR5 typing data of 1,333,035 potential hematopoietic stem cell donors enlisted with three national DKMS donor centers. Allele and genotype frequencies were determined for 87 countries of origin as self-assessed by the donors. CCR5-Δ32 allele frequencies ranged from 16.4% in the Norwegian sample to 0 in donors from Ethiopia. The highest CCR5-Δ32/Δ32 genotype frequency was found in the sample from the Faroe Islands (2.3%), whereas in 27 samples, predominantly of donors from Africa, Asia and South America, none of the individuals carried this genotype. The characteristic CCR5-Δ32 allele frequency decline from Northern to Southeastern Eurasia supports findings of earlier studies. With available HLA haplotype frequency information for the patient’s ethnicity, our data allows upfront estimation of the probability that an HLA-matched donor with CCR5-Δ32/Δ32 genotype can be found for a patient in need of hematopoietic stem cell transplantation.