Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions.

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

Light-Activated Staudinger-Bertozzi Ligation within Living Animals.

The ability to regulate small molecule chemistry in vivo will enable new avenues of exploration in imaging and pharmacology. However, realization of these goals will require reactions with high specificity and precise control. Here we demonstrate photocontrol over the highly specific Staudinger-Bertozzi ligation in vitro and in vivo. Our simple approach, photocaging the key phosphine atom, allows for the facile production of reagents with photochemistry that can be engineered for specific applications. The resulting compounds, which are both stable and efficiently activated, enable the spatial labeling of metabolically introduced azides in vitro and on live zebrafish.

Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway.

Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked ß-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

Altering adenoviral tropism via click modification with ErbB specific ligands.

Methods for targeting oncolytic viruses can increase efficacy and accelerate development. Genetic engineering, the predominant method for changing vector tropism, is limited in scope and often represents the bottleneck for vector development. Metabolic incorporation of an unnatural azido sugar, O-GlcNAz, at a specific site on the adenoviral surface allows chemoselective attachment of affibodies for Her2 or EGF receptors. Modification with these high-affinity, high-selectivity proteins is straightforward and readily generalizable, demonstrates minimal impact on virus physiology, and affords significant increases in gene delivery to cancer cells. As a result, this method has significant potential to increase the efficacy of next-generation viral vectors.

Targeted and armed oncolytic adenovirus via chemoselective modification.

Oncolytic adenoviruses (Ads) are an emerging alternative therapy for cancer; however, clinical trial have not yet demonstrated sufficient efficacy. When oncolytic Ads are used in combination with taxoids a synergistic increase in both cytotoxicity and viral replication is observed. In order to generate a next generation oncolytic adenovirus, virion were physically conjugated to a highly potent taxoid, SB-T-1214, and a folate targeting motif. Conjugation was enabled via the metabolic incorporation of non-canonical monosaccharides (O-GlcNAz) and amino acids (homopropargylglycine), which served as sites for chemoselective modification.

Compartment-Specific Labeling of Bacterial Periplasmic Proteins by Peroxidase-Mediated Biotinylation.

The study of the bacterial periplasm requires techniques with sufficient spatial resolution and sensitivity to resolve the components and processes within this subcellular compartment. Peroxidase-mediated biotinylation has enabled targeted labeling of proteins within subcellular compartments of mammalian cells. We investigated whether this methodology could be applied to the bacterial periplasm. In this study, we demonstrated that peroxidase-mediated biotinylation can be performed in mycobacteria and Escherichia coli. To eliminate detection artifacts from natively biotinylated mycobacterial proteins, we validated two alternative labeling substrates, tyramide azide and tyramide alkyne, which enable biotin-independent detection of labeled proteins. We also targeted peroxidase expression to the periplasm, resulting in compartment-specific labeling of periplasmic versus cytoplasmic proteins in mycobacteria. Finally, we showed that this method can be used to validate protein relocalization to the cytoplasm upon removal of a secretion signal. This novel application of peroxidase-mediated protein labeling will advance efforts to characterize the role of the periplasm in bacterial physiology and pathogenesis.

Metabolic labeling of glycans with azido sugars for visualization and glycoproteomics.

The staggering complexity of glycans renders their analysis extraordinarily difficult, particularly in living systems. A recently developed technology, termed metabolic oligosaccharide engineering, enables glycan labeling with probes for visualization in cells and living animals, and enrichment of specific glycoconjugate types for proteomic analysis. This technology involves metabolic labeling of glycans with a specifically reactive, abiotic functional group, the azide. Azido sugars are fed to cells and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged, either ex vivo or in vivo, using one of two azide-specific chemistries: the Staudinger ligation, or the strain-promoted [3+2] cycloaddition. These reactions can be used to tag glycans with imaging probes or epitope tags, thus enabling the visualization or enrichment of glycoconjugates. Applications to noninvasive imaging and glycoproteomic analyses are discussed.

Surface modification via strain-promoted click reaction facilitates targeted lentiviral transduction.

As a result of their ability to integrate into the genome of both dividing and non-dividing cells, lentiviruses have emerged as a promising vector for gene delivery. Targeted gene transduction of specific cells and tissues by lentiviral vectors has been a major goal, which has proven difficult to achieve. We report a novel targeting protocol that relies on the chemoselective attachment of cancer specific ligands to unnatural glycans on lentiviral surfaces. This strategy exhibits minimal perturbation on virus physiology and demonstrates remarkable flexibility. It allows for targeting but can be more broadly useful with applications such as vector purification and immunomodulation.

Chemoselective modification of viral surfaces via bioorthogonal click chemistry.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development. Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction. Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible. The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically or analytically, as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness, (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access. In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only. In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein - strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Chemoselective modification of viral proteins bearing metabolically introduced "clickable" amino acids and sugars.

The inherent difficulty of performing chemical modifications of proteins in a truly site-specific fashion is often compounded by the need to work within complex biological settings. In order to alleviate this complication, targets can be "prelabeled" metabolically with unnatural residues, which allow access to highly selective bioorthogonal reactions. Due to their small size, permissibility within biosynthetic pathways and access to reactions with high specificity, azides provide excellent bioorthogonal handles. This two-step labeling process is emerging as a highly effective means to modify therapeutic proteins. In this chapter, we take this strategy a step further and apply chemoselective ligation to remodel the surfaces of adenoviruses. Despite the large number of ongoing clinical trials involving these complex mammalian viruses, new methods for their facile, flexible surface modification are necessary to drive the development of next-generation therapeutics. Here we demonstrate the modification of azides on adenoviral surfaces via a straightforward chemoselective protocol based on copper-assisted "click" chemistry. This method provides access to a wide array of effector functionalities without sacrificing infectivity.

Chemoselective modification of proteins: hitting the target.

The use of synthetic molecules to modulate and track biological events is a central component of chemical biology. As a result, the precise, covalent modification of biomolecules is a key goal for this field. Several strategies have emerged that allow specific tailoring of polypeptides through either endogenous residues or introduced functionality. This tutorial review discusses these recent advances in the context of in vitro and in vivo applications.

Introducing genetically encoded aldehydes into proteins.

Methods for introducing bioorthogonal functionalities into proteins have become central to protein engineering efforts. Here we describe a method for the site-specific introduction of aldehyde groups into recombinant proteins using the 6-amino-acid consensus sequence recognized by the formylglycine-generating enzyme. This genetically encoded 'aldehyde tag' is no larger than a His(6) tag and can be exploited for numerous protein labeling applications.

A designed phenylalanyl-tRNA synthetase variant allows efficient in vivo incorporation of aryl ketone functionality into proteins.

Incorporation of non-natural amino acids into proteins in vivo expands the scope of protein synthesis and design. p-Acetylphenylalanine was incorporated into recombinant dihydrofolate reductase (DHFR) in Escherichia coli via a computationally designed mutant form of the phenylalanyl-tRNA synthetase of the host. DHFR outfitted with ketone functionality can be chemoselectively ligated with hydrazide reagents under mild conditions.