Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors Export

@article{citeulike:4817516, abstract = {We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR ( α / β 19′GGGU/ γ / δ ) mRNAs. We measured fluorescence from oocytes expressing nAChR β 19′Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/ μ m 2 ), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR β 19′Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR β 19′Lys(BODIPYFL) receptors labeled with α -bungarotoxin monoconjugated with Alexa488 ( α BtxAlexa488). The nAChR has two α Btx binding sites, and puncta containing the Lys(BODIPYFL) labeled with α BtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the γ -subunit M3-M4 loop, which confirmed our nAChR β 19′Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR β 19′Lys(BODIPYFL).}, author = {Pantoja, R. and Rodriguez, E. and Dibas, M. and Dougherty, D. and Lester, H.}, citeulike-article-id = {4817516}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/j.bpj.2008.09.034}, citeulike-linkout-1 = {http://linkinghub.elsevier.com/retrieve/pii/S000634950800012X}, doi = {10.1016/j.bpj.2008.09.034}, issn = {00063495}, journal = {Biophysical Journal}, keywords = {bioconjugation, biophysics, sms}, month = {January}, number = {1}, pages = {226--237}, posted-at = {2009-06-11 23:59:46}, priority = {2}, title = {Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors}, url = {http://dx.doi.org/10.1016/j.bpj.2008.09.034}, volume = {96}, year = {2009} }

TY - JOUR ID - citeulike:4817516 L3 - citeulike-article-id:4817516 N2 - We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR ( α / β 19′GGGU/ γ / δ ) mRNAs. We measured fluorescence from oocytes expressing nAChR β 19′Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/ μ m 2 ), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR β 19′Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR β 19′Lys(BODIPYFL) receptors labeled with α -bungarotoxin monoconjugated with Alexa488 ( α BtxAlexa488). The nAChR has two α Btx binding sites, and puncta containing the Lys(BODIPYFL) labeled with α BtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the γ -subunit M3-M4 loop, which confirmed our nAChR β 19′Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR β 19′Lys(BODIPYFL). IS - 1 JF - Biophysical Journal SN - 00063495 EP - 237 TI - Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors VL - 96 SP - 226 KW - bioconjugation KW - biophysics KW - sms AU - Pantoja, R AU - Rodriguez, E AU - Dibas, M AU - Dougherty, D AU - Lester, H PY - 2009/01/07/ UR - http://dx.doi.org/10.1016/j.bpj.2008.09.034 ER -