Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

Zhang, Zhiwen; Alfonta, Lital; Tian, Feng; Bursulaya, Badry; King, David S.; Schultz, Peter G.

doi:10.1073/pnas.0307029101

Cited by 129 publications

(107 citation statements)

References 35 publications

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“…[6][7][8][9] The first absorption band of 5-hydroxytryptophan is significantly longer than that of tryptophan, allowing easy discrimination of the signal. There have been some qualitative experimental studies using 5-hydroxytryptophan, such as with bacteriophage λ-cI repressor, in which 5-hydroxytryptophan replaced the three wild-type tryptophans.…”

Section: Introductionmentioning

confidence: 99%

“…These approaches typically rely on the use of extrinsic labeling with spectroscopic probes, as biomolecules possess few natural moieties with useful optical properties. [1][2][3][4][5][6][7][8][9] In membranes and cells, for example, it is necessary to utilize spectroscopic labels directed at useful properties. 10 These often include measurement of shifts of the emission or excitation spectra that yield desirable information about some cellular property such as membrane fluidity, ion concentration, etc.…”

Section: Introductionmentioning

confidence: 99%

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Calculating the Fluorescence of 5-Hydroxytryptophan in Proteins

et al. 2009

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5-Hydroxytryptophan is a non-natural amino acid that has attracted a lot of recent interest as a fluorescent probe of protein structure, dynamics, and function. We have investigated its fluorescence in various protein environments, using a decoupled quantum mechanics/molecular mechanics approach. Classical, all-atom molecular dynamics simulations of several proteins containing single tryptophans were performed for both the wild-type and the 5-hydroxy derivatives. The excited state of the fluorophore was described using parameters from complete active space self-consistent field calculations. Time-dependent density functional theory calculations on 5-hydroxytryptophan and a significant portion of its explicit immediate surrounding environment, sampled by the simulations, show that the emission energies of 5-hydroxytryptophan shift, depending on the strength of hydrogen bonding and π-π stacking interactions. This quantitative description of how the fluorescence responds to different protein environments should enhance the insight that fluorescence studies using 5-hydroxytryptophan can provide at a molecular level.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Calculating the Fluorescence of 5-Hydroxytryptophan in Proteins

et al. 2009

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“…Introduction of a photoactivatable group into specific positions was achieved using suppressor tRNAs for translation in vitro 10 and in vivo 11,12 . Using engineered 'orthogonal' pairs of tRNAs and aminoacyl tRNA synthetases (aaRSs), various unnatural amino acids now can be incorporated into specific sites of proteins in cultured mammalian cells [13][14][15] . Whereas this method is well-suited for the production of engineered proteins, its application for global detection of protein-protein interactions in cell biology is limited by the complexity of the modified components (that is, tRNA, aaRS, cDNA and unnatural amino acid) required and by the fact that only a single position in a single protein will bear the photoreactive group.…”

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confidence: 99%

Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells

2005

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Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-crosslinking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.

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“…Most prokaryotic tRNAs do not contain these consensus sequences and are poorly transcribed in eukaryotic cells. Careful selection of a prokaryotic tRNA that contains consensus A-and B-box elements, or are permissive to mutation in order to include them, has been used successfully (Sakamoto et al, 2002;Zhang et al, 2004). However, the strict recognition elements of aaRSs for tRNAs often prevents modifications of the tRNA sequence to introduce these elements.…”

Section: Stop Codonmentioning

confidence: 99%