Site‐Specific Labelling of Native Mammalian Proteins for Single‐Molecule FRET Measurements

Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single‐molecule FRET measurements allow exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome, as a method for the site‐specific coupling of organic dyes into native, non‐recombinant mammalian proteins is lacking. We address this issue showing the site‐specific engineering of fluorescent dyes into human proteins on the basis of bioorthogonal reactions. We show the applicability of the method to study functional and post‐translationally modified proteins on the single‐molecule level, among them the hitherto inaccessible human Argonaute 2.     

Site‐Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne–Azide Cycloaddition Reactions

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine‐reactive probes that target surface‐exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)‐binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site‐specifically labeled product. Z₃₂BPA was synthesized by solid‐phase peptide synthesis and further functionalized to give alkyne‐Z₃₂BPA and azide‐Z₃₂BPA for Cu^I‐catalyzed cycloaddition, as well as DBCO‐Z₃₂BPA for Cu‐free strain‐promoted cycloaddition. The Z₃₂BPA variants were conjugated to the human IgG1 antibody trastuzumab and site‐specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2‐expressing cells in immunofluorescence microscopy.     

A Genetically Encoded Diazirine Analogue for RNA–Protein Photo-crosslinking

Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA–protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA-binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA–protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA-binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA-binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine-based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA–protein interactions.     

Native Chemical Ligation Combined with Desulfurization and Deselenization: A General Strategy for Chemical Protein Synthesis

Of the many approaches proposed to generalize the native chemical ligation approach for protein synthesis, the simple procedure of global desulfurization of peptide thiols has become the most widely adopted. In this review, the development of the native ligation–desulfurization strategy is described, focusing on the conversion of Cys to Ala following ligation at N-terminal Cys residues. Subsequent variations on this theme have broadened the scope to other natural amino acids including Phe, Leu, Val, and Lys, and even non-native peptide linkages such as isopeptide bonds on lysine side chains. Using insights from both selenocysteine–peptide side reactions and radical initiated desulfurization procedures, a new method for the selective deselenization of peptides containing both selenocysteine and cysteine residues has been developed. Together, these approaches represent a robust and flexible methodology for the synthesis of complex polypeptides without the use of protecting groups.     

Characterisation of Photoaffinity‐Based Chemical Probes by Fluorescence Imaging and Native‐State Mass Spectrometry

Chemical probes are small‐molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer‐associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug‐resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein–probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein–probe binding, and covalent protein–probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure–activity relationships to support the development of optimum chemical probes.     

Dual Unnatural Amino Acid Incorporation and Click‐Chemistry Labeling to Enable Single‐Molecule FRET Studies of p97 Folding

Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multidomain AAA+ chaperone. Given the complex architecture of p97, single‐molecule (sm) FRET would be a powerful tool for studying folding while avoiding ensemble averaging. However, dual site‐specific labeling of such a large protein for smFRET is a significant challenge. Here, we address this issue by using bioorthogonal azide–alkyne chemistry to attach an smFRET dye pair to site‐specifically incorporated unnatural amino acids, allowing us to generate p97 variants reporting on inter‐ or intradomain structural features. An initial proof‐of‐principle set of smFRET results demonstrated the strengths of this labeling method. Our results highlight this as a powerful tool for structural studies of p97 and other large protein machines.     

An Azide‐Modified Nucleoside for Metabolic Labeling of DNA

Metabolic incorporation of azido nucleoside analogues into living cells can enable sensitive detection of DNA replication through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) and strain‐promoted azide–alkyne cycloaddition (SPAAC) “click” reactions. One major limitation to this approach is the poor chemical stability of nucleoside derivatives containing an aryl azide group. For example, 5‐azido‐2′‐deoxyuridine (AdU) exhibits a 4 h half‐life in water, and it gives little or no detectable labeling of cellular DNA. In contrast, the benzylic azide 5‐(azidomethyl)‐2′‐deoxyuridine (AmdU) is stable in solution at 37 °C, and it gives robust labeling of cellular DNA upon addition of fluorescent alkyne derivatives. In addition to providing the first examples of metabolic incorporation into and imaging of azide groups in cellular DNA, these results highlight the general importance of assessing azide group stability in bioorthogonal chemical reporter strategies.     

Functional Modification of Fibronectin by N‐Terminal FXIIIa‐Mediated Transamidation

A straightforward strategy is presented for the site‐specific incorporation of fluorophores or reactive probes into the extracellular matrix (ECM) protein fibronectin (Fn) by using the enzyme‐catalyzed transamidation by activated factor XIII. Characterization by SDS‐PAGE, western blotting, absorption measurements, mass spectrometry, and stepwise photobleaching for labeling quantification at the single‐molecule level showed that the labeling was efficient and restricted to the N‐terminal tails. The introduction of labels did not interfere with Fn fibrillogenesis, as verified by the incorporation of fluorescently labeled Fn into ECM and manually pulled Fn fibers. Site‐specific incorporation of an azide was used to create a template for bioorthogonal click chemistry reactions in a second bioconjugation step, thus offering versatile modification and application possibilities in the context of matrix biology and tissue engineering.     

Amino Acid‐Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology

Current solution NMR techniques enable structural investigations of proteins in molecular particles with sizes up to several hundred kDa. However, the large molecular weight of proteins in such systems results in increased numbers of NMR signals, and the resulting spectral overlap typically imposes limitations. For multidomain proteins, segmental isotope labeling of individual domains facilitates the spectral interpretation by reducing the number of signals, but for large domains with small signal dispersion, signal overlap can persist. To overcome limitations arising from spectral overlap, we present a strategy that combines cell‐free expression and ligation of the expressed proteins to produce multidomain proteins with selective amino acid‐type labeling in individual domains. The bottleneck of intrinsically low cell‐free expression yields of precursor molecules was overcome by introducing new fusion constructs that allowed milligram production of ligation‐competent domains labeled in one or multiple amino acid types. Ligation‐competent unlabeled partner domains were produced in vivo, and subsequent domain ligation was achieved by using an on‐column strategy. This approach is illustrated with two multidomain RNA‐binding proteins, that is, the two C‐terminal RNA‐recognition motifs of the human polypyrimidine tract‐binding protein, and two highly homologous helix–turn–helix domains of the human glutamyl‐prolyl‐tRNA synthetase.     

Hydrophilic trans‐Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling

Introduction of bioorthogonal functionalities (e.g., trans‐cyclooctene‐TCO) into a protein of interest by site‐specific genetic encoding of non‐canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine‐functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours‐long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO‐ncAAs. One derivative, DOTCO‐lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.     

Exploiting the substrate tolerance of farnesyltransferase for site-selective protein derivatization

The site-selective modification of proteins with a functional group is an important biochemical technique, but covalent attachment of a desired group to a chosen site is complicated by the reactivity of other amino acid side chains, often resulting in undesired side reactions. One potential solution to this problem involves exploiting the activity of protein-modifying enzymes that recognize a defined protein sequence. Protein farnesyltransferase (FTase) covalently attaches an isoprenoid moiety to a cysteine unit in the context of a short C-terminal sequence that can be easily grafted onto recombinant proteins. Here we describe the synthesis of four phosphoisoprenoids functionalized with biotin, azide, or diene groups. These phosphoisoprenoids bound to FTase with affinities comparable to that of the native substrate. With the exception of the biotin-functionalized analogue, all the phosphoisoprenoids generated could be transferred to peptide and protein substrates by FTase. Unlike proteins modified with farnesyl moieties, Ypt7 prenylated with (2E,6E)-8-(azidoacetamido)-3,7-dimethylocta-2,6-dienyl groups did not oligomerize and showed no detectable increase in hydrophobicity. To assess the suitability of the functionalized isoprenoids for protein modifications they were further derivatized, both by Diels-Alder cycloaddition with 6-maleimidohexanoic acid and by Staudinger ligation with a phosphine. We demonstrate that the Staudinger ligation proceeds more rapidly and is more efficient than the Diels-Alder cycloaddition. Our data validate the use of FTase as a protein-modification tool for biochemical and biotechnological applications.