Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels–Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of <i>trans</i>-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells

Bifunctional Fatty Acid Chemical Reporter for Analyzing S‑Palmitoylated Membrane Protein–Protein Interactions in Mammalian Cells

Studying the functions of S-palmitoylated proteins in cells can be challenging due to the membrane targeting property and dynamic nature of protein S-palmitoylation. New strategies are therefore needed to specifically capture S-palmitoylated protein complexes in cellular membranes for dissecting their functions <i>in vivo</i>. Here we present a bifunctional fatty acid chemical reporter, x-alk-16, which contains an alkyne and a diazirine, for metabolic labeling of S-palmitoylated proteins and photo-cross-linking of their involved protein complexes in mammalian cells. We demonstrate that x-alk-16 can be metabolically incorporated into known S-palmitoylated proteins such as H-Ras and IFITM3, a potent antiviral protein, and induce covalent cross-linking of IFITM3 oligomerization as well as its specific interactions with other membrane proteins upon in-cell photoactivation. Moreover, integration of x-alk-16-induced photo-cross-linking with label-free quantitative proteomics allows identification of new IFITM3 interacting proteins

Additional file 5: of The PhyR homolog RSP_1274 of Rhodobacter sphaeroides is involved in defense of membrane stress and has a moderate effect on RpoE (RSP_1092) activity

Northern blot analysis of Pos19. Cultures were treated with t-BOOH and samples taken at time point 0 and 7 min. Pos19 bands were normalized to the 5S rRNA and the calculated fold change is indicated. (PDF 669 kb

Additional file 3: of The PhyR homolog RSP_1274 of Rhodobacter sphaeroides is involved in defense of membrane stress and has a moderate effect on RpoE (RSP_1092) activity

Growth of the Rhodobacter sphaeroides wild type 2.4.1 and various mutant strains after heat shock. Cultures were grown at 32 °C to exponential phase in microaerobic conditions and diluted to OD660 of 0.1. For each strain 5 μl of diluted culture were spread on agar plates and incubated under the indicated temperature in the dark. The agar plates incubated at 42 °C were shifted to 32 °C after 24 h. (PDF 791 kb

Isolating a Trimer Intermediate in the Self-Assembly of E2 Protein Cage

Understanding the self-assembly mechanism of caged proteins provides clues to develop their potential applications in nanotechnology, such as a nanoscale drug delivery system. The E2 protein from Bacillus stearothermophilus, with a virus-like caged structure, has drawn much attention for its potential application as a nanocapsule. To investigate its self-assembly process from subunits to a spherical protein cage, we truncate the C-terminus of the E2 subunit. The redesigned protein subunit shows dynamic transition between monomer and trimer, but not the integrate 60-mer. The results indicate the role of the trimer as the intermediate and building block during the self-assembly of the E2 protein cage. In combination with the molecular dynamics simulations results, we conclude that the C-terminus modulates the self-assembly of the E2 protein cage from trimer to 60-mer. This investigation elucidates the role of the intersubunit interactions in engineering other functionalities in other caged structure proteins

Additional file 1: of The PhyR homolog RSP_1274 of Rhodobacter sphaeroides is involved in defense of membrane stress and has a moderate effect on RpoE (RSP_1092) activity

Strains and plasmids (Table S1), Oligodeoxynucleotides (Table S2) used in this study. (PDF 163 kb

Comparisons of the DEGs data and qRT-PCR results.

<p>6.47*: up-regulated at larval stage (P value<0.001);</p><p>−5.39**: down-regulated at adult stage (P value<0.001);</p><p><i>TK</i> (control) ***: non-DEG.</p

Characteristic analysis of the homology search of ESTs against the nr database.

<p>(A) Identity distribution of the top BLAST hits for each sequence. (B) Species distribution is shown as a percentage of the total homologous sequences with an E-value of at least 1.0E⁻⁵. The first hit of each sequence was used for analysis. Homo: <i>Homo sapiens</i>; Rat: <i>Rattus norvegicus</i>.</p

Assembled contig length distribution of the <i>B.dorsalis</i> transcriptome.

<p>The x-axis indicates contig size and the y-axis indicates the number of contigs of each size.</p