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.
Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-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.
Unnatural amino acids with useful chemical functionality can replace phenylalanine in bacterial proteins. Coexpression of a promiscuous phenylalanine‐tRNA synthetase mutant enables the synthesis of target proteins bearing iodophenyl, cyanophenyl, ethynylphenyl, azidophenyl, and pyridyl groups (see general structures). Proteins incorporating the analogues have a range of potential applications, including Pd‐mediated conjugation (R=CCH), photoaffinity labeling (R=N3), X‐ray phasing (R=I), and novel metal coordination (R=pyridyl).
Highly sensitive: The azido analogue of methionine, azidohomoalanine (see picture), is shown to be a sensitive IR probe of protein structure, folding, and electrostatics, as demonstrated for ribosomal protein NTL9. It can be readily incorporated in to proteins, and the azido frequency is significantly blue‐shifted in the thermally unfolded state.
Control of the spatial arrangement of proteins on surfaces is essential in a number of emerging technologies, including protein microarrays, biosensors, 1 tissue engineering, and regenerative medicine. 2 Patterning is also a powerful tool in cell biology, wherein cell arrays are used to elucidate the factors that mediate migration, proliferation, and cell-cell interactions. 3 Although photolithography holds a preeminent place as a patterning method in the microelectronics industry, optical lithography of proteins has been hampered by the need either to use traditional chemical photoresists or to modify proteins chemically by attachment of photoreactive functional groups; both methods can compromise protein function. 4Production of a protein "photoresist" without the need for post-translational chemical modification would require an intrinsically photoreactive protein. Recently, the incorporation of photo-reactive, non-canonical amino acids into proteins via site-specific 5 and residuespecific techniques has been reported. 6 Here we describe the microbial expression of artificial proteins bearing the photosensitive non-canonical amino acid para-azidophenylalanine (pN 3 Phe). The recombinant proteins, designated artificial extracellular matrix proteins with aryl azides (aECM-N 3 ), belong to a family of engineered proteins designed to exhibit mechanical properties similar to those of native elastins 7 and to support adhesion of mammalian cells through cell-binding domains (CS5 or RGD) derived from fibronectin ( Fig 1A). 8 These proteins can be crosslinked efficiently upon irradiation at 365 nm. The physical properties of the crosslinked films can be controlled by changing the pN 3 Phe content, and thin films can be patterned on surfaces via photolithographic techniques. We demonstrate the utility of the method by creating cell arrays through selective cell attachment to photolithographically prepared protein patterns. aECM-N 3 variants were expressed in Escherichia coli cultures supplemented with pN 3 Phe (Supporting Information). Incorporation of pN 3 Phe into the recombinant proteins relies on activation of the photosensitive amino acid by the phenylalanyl-tRNA synthetase (PheRS) of the bacterial expression host. The PheRS used for this study was a previously characterized mutant with relaxed substrate specificity. 9 This method results in statistical decoding of phenylalanine (Phe) codons placed at regular intervals in the coding sequence. 9 Proteins were expressed in a Phe-auxotrophic E. coli strain and purified by exploiting the temperaturedependent phase behavior of proteins that contain elastin-like repeats. 10 Incorporation efficiency was determined by integration of the aromatic proton signals in the 1 H NMR spectra of the purified proteins; the extent of Phe replacement varied from 13% to 53%, depending on the concentration of pN 3 Phe in the expression medium (Supporting Information). Understanding the response of the photoreactive protein to irradiation is crucial for highresolution pattern formation....
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.
The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.
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