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, 24 (5), 548-550

"Expand and Click": A New Method for Labeling HIV-1 Envelope Glycoproteins

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"Expand and Click": A New Method for Labeling HIV-1 Envelope Glycoproteins

Melissa V Fernandez et al. Cell Chem Biol.

Abstract

In this issue of Cell Chemical Biology, Sakin et al. (2017) investigate the nanoscale behavior of the HIV-1 envelope (Env) glycoprotein complex by using genetic code expansion, bioorthogonal amino acids, synthetic dyes, and click chemistry. This minimally invasive approach allows the measurement of native Env cellular distribution and dynamics.

Figures

Figure 1.
Figure 1.. A Site-Specific, Covalent Labeling Strategy for the HIV-1 Env Glycoprotein Complex Expressed on the Plasma Membrane of Living Cells
(A) Translational machinery orthogonal to the host machinery is introduced into the target cell in the presence of ncAA (in this example the ncAA is BCN-Lys) to suppress amber stop codons. The tRNA synthase is engineered to specifically aminoacylate tRNACUA with the ncAA, resulting in incorporation of the ncAA into the growing polypeptide chain wherever there is an amber stop codon. (B) Translation in suppressed cells results in a mixture of full-length, ncAA-labeled, plasma membrane-associated Env and truncated, cytoplasmic Env. Supplementation of cellular media with H-Tet-Cy5 or H-Tet-KK114 (for fluorescence recovery after photobleaching or STED analysis, respectively) results in a click labeling reaction between ncAA and tetrazine to produce a covalently labeled Env trimer on membrane-associated, full-length Env trimers. (c) Env mobility and association with Gag-GFP can then be measured.

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