Dan Groff

A Facile System for Encoding Unnatural Amino Acids in Mammalian Cells

A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA-aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.

Evolution of Amber Suppressor tRNAs for Efficient Bacterial Production of Proteins Containing Nonnatural Amino Acids

Open in a separate window Regions of the M. jannaschii tyrosyl tRNACUA thought to interact with elongation factor Tu were randomized, and the resulting tRNA libraries were subjected to in vitro evolution. The tRNAs identified resulted in significantly improved unnatural amino acid-containing protein yields. In some cases, the degree of improvement varied in an amino acid-dependent manner.

A Genetically Encoded ε‐N‐Methyl Lysine in Mammalian Cells

The posttranslational methylation of lysine modulates the activity, stability, localization and biomolecular interactions of many eukaryotic proteins. For example, monomethylation of lysine 372 in the mammalian tumor suppressor p53 has been shown to affect protein stability and localization[1]. Protein methylation plays a particularly important role in gene expression due to its involvement in the histone code, in which specific modifications to histone proteins modulate the transcriptional status of specific genes. Methylation of distinct histone lysine residues has been correlated with both transcriptional activation and repression depending on the lysine modified.[2] To better understand the functional consequences of lysine methylation, methods are needed to generate proteins with defined methylation status both in vitro and in living cells.

Efforts Toward the Direct Experimental Characterization of Enzyme Microenvironments: Tyrosine100 in Dihydrofolate Reductase†

State secrets: Site-specific deuteration and FTIR studies reveal that Tyr100 in dihydrofolate reductase plays an important role in catalysis, with a strong electrostatic coupling occurring between Tyr100 and the charge that develops in the hydride-transfer transition state (see picture, NADP(+) purple, Tyr100 green). However, relaying correlated motions that facilitate catalysis from distal sites of the protein to the hydride donor may also be involved.

A New Strategy to Photoactivate Green Fluorescent Protein

Photoactivatable fluorescent proteins have become an important addition to the set of molecular probes used to understand cellular function. Known as molecular highlighters, their fluorescence is switched on by irradiation, thereby enabling non-invasive tracking of protein trafficking and dynamics.[1] They are also the basis for the imaging technique PhotoActivated Light Microscopy (PALM)[2] in which multiple emitted photons are observed from individual active fluorophores which are sequentially activated from a large pool of inactive proteins and then photobleached. By locating the center of the point spread function for these emitted photons, it is possible to determine the location of the active fluorophore at better resolution than the theoretical diffraction limit. Previous GFP mutants exhibiting photomodulatory behaviour have been reported including Kaede[3] and KikGR[4] whose emission wavelength change irreversibly, Dronpa[5] whose fluorescence can be reversibly activated with light, and photoactivatable GFP (paGFP)[1], whose excitation wavelength can be irreversibly changed with light. For Kaede, KikGR, and Dronpa, the precise three dimensional structure of the fluorophore must be preserved to maintain photoswitching behavior[4, 6]. In paGFP, the shift in excitation wavelength is mediated by a light dependent decarboxylation of Glu222.[1, 7] The loss of this carboxyl group is believed to cause reorientation of an internal hydrogen bond network, which changes the protonation state of the fluorophore and leads to an irreversible shift in the excitation maximum from 397 nm to 475 nm. This mechanism of GFP photoactivation requires the preservation of active site residues His203, His148, Ser205 and Glu222. Unfortunately the sequence restrictions necessary to maintain photomodulatory behavior are not always compatible with the mutations necessary to produce other fluorescent protein variants including YFP[8, 9] and RFP[10]. In addition, Kaede, KikGR and paGFP exist in two forms with differing excitation and emission wavelengths, precluding the simultaneous use of these wavelengths for other purposes. To address these problems, we have developed a general strategy for photoactivating GFP based upon unnatural amino acid mutagenesis[11] with the photocaged tyrosine analog o-nitrobenzyl-O-tyrosine (ONBY).[12] Replacing the fluorophore tyrosine 66 with ONBY yields a GFP molecule that is nonfluorescent as observed for other o-nitrobenzyl appended fluorophores including fluoresceine,[13] texas red,[14] and quantum dots.[15] An earlier unnatural variant of GFP was produced using a similar strategy, however, the high preirradiation background fluorescence and low protein yield made this unsuitable for use as a molecular marker.[16] Fast, time resolved UV-Vis spectroscopy measurements indicate that the fluorescence quenching likely occurs through Photo-induced Electron Transfer (PET). Irradiation at 365 nm is sufficient to remove the o-nitrobenzyl group and restore fluorescence to GFP Tyr66→ONBY (GFP66ONBY).