Site-specific labeling of proteins with NMR-active unnatural amino acids

Jones, David H.; Cellitti, Susan E.; Hao, Xueshi; Zhang, Qiong; Jahnz, Michael; Summerer, Daniel; Schultz, Peter G.; Uno, Tetsuo; Geierstanger, Bernhard H.

doi:10.1007/s10858-009-9365-4

Cited by 82 publications

(78 citation statements)

References 105 publications

(123 reference statements)

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“…Unfortunately, incorporation of this UAA triggers metal-mediated protein oligomerization, 61 which is also a potential problem associated with bipyridyl-alanine 62 after binding paramagnetic Co(II). 63 In our hands, the Cu(I)-catalyzed click reactions were usually accompanied by protein precipitation during the ligation reaction, but only the p75 ICD samples continued to precipitate during NMR measurements.…”

Section: +mentioning

confidence: 99%

Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

et al. 2013

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Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclenbased paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide−alkyne cycloaddition to a genetically encoded p-azido-L-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.

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Section: +mentioning

confidence: 99%

Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

et al. 2013

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“…Site-specific labeling of proteins can be accomplished by using genetic code expansion technology [112]. For example, 13 C/ 15 N-labeled p-methoxyphenylalanine (Fig.…”

Section: Residue Labeling For Nuclear Magnetic Resonancementioning

confidence: 99%

Biochemical analysis with the expanded genetic lexicon

2012

Anal Bioanal Chem

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The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods.

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“…Because wild-type aaRS does not usually recognize a NCAA to be used for site-specific incorporation, the active sites of aaRSs are often redesigned to recognize a NCAA, which is a typical protein engineering topic. Indeed, rational design and directed evolution of aaRS have been frequently employed to tailor the substrate specificity of aaRS towards various NCAAs [16,25,26,[72][73][74][75]. In particular, Schultz's group [4] developed a powerful aaRS library screening method to identify aaRS variants specific for each NCAA.…”

Section: Site-specific Incorporation Of Ncaas Into Enzymesmentioning

confidence: 99%

“…The nitrile group of p-cyanophenylalanine has been used as a fluorescence and infrared probe to study ligand binding to myoglobin, folding of ribosomal protein L9, and aggregation of islet amyloid polypeptide [13][14][15]. Incorporating NCAA can also facilitate NMR spectroscopic studies of proteins (reviewed in [16]). In particular, labeling a protein with a NCAA containing a 19 F atom is a very popular method to characterize protein structural changes by NMR spectroscopy [17][18][19][20][21][22].…”

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confidence: 99%

Manipulation of enzyme properties by noncanonical amino acid incorporation

Zheng

Kwon

2011

Biotechnology Journal

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Since wild-type enzymes do not always have the properties needed for various applications, enzymes are often engineered to obtain desirable properties through protein engineering techniques. In the past decade, complementary to the widely used rational protein design and directed evolution techniques, noncanonical amino acid incorporation (NCAAI) has become a new and effective protein engineering technique. Recently, NCAAI has been used to improve intrinsic functions of proteins, such as enzymes and fluorescent proteins, beyond the capacities obtained with natural amino acids. Herein, recent progress on improving enzyme properties through NCAAI in vivo is reviewed and the challenges of current approaches and future directions are also discussed. To date, both NCAAI methods-residue- and site-specific incorporation-have been primarily used to improve the catalytic turnover number and substrate binding affinity of enzymes. Numerous strategies used to minimize structural perturbation and stability loss of a target enzyme upon NCAAI are also explored. Considering the generality of NCAAI incorporation, we expect its application could be expanded to improve other enzyme properties, such as substrate specificity and solvent resistance in the near future.

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Site-specific labeling of proteins with NMR-active unnatural amino acids

Cited by 82 publications

References 105 publications

Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

Biochemical analysis with the expanded genetic lexicon

Manipulation of enzyme properties by noncanonical amino acid incorporation

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