Bioincorporation of telluromethionine into proteins: a promising new approach for X-ray structure analysis of proteins 1 1Edited by K. Nagai

Budiša, Nediljko; Karnbrock, Wilhelm; Steinbacher, Stefan; Humm, A.; Prade, Lars; Neuefeind, Torsten; Moröder, Luis; Huber, Robert

doi:10.1006/jmbi.1997.1132

Cited by 74 publications

(73 citation statements)

References 17 publications

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“…When successful, this substitution provides a strong anomalous signal and therefore significant phasing power (Budisa et al, 1997).…”

Section: Not Enough Methionines?mentioning

confidence: 99%

Selenium incorporation using recombinant techniques

Walden

2010

Acta Crystallogr D Biol Crystallogr

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Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

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“…When successful, this substitution provides a strong anomalous signal and therefore significant phasing power (Budisa et al, 1997).…”

Section: Not Enough Methionines?mentioning

confidence: 99%

Selenium incorporation using recombinant techniques

Walden

2010

Acta Crystallogr D Biol Crystallogr

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“…Moreover, it has been shown that mutation of the Met residues in CaM to other amino acids, even to the related hydrophobic Leu residues, may impair the CaM activation of some target proteins~Zhang et al, 1994b;Chin & Means, 1996;Chin et al, 1997b;Edwards et al, 1998!. Substitution of amino acids in proteins with unnatural amino acid analogs is a relatively old approach in protein chemistry~for reviews, see Richmond, 1962;Hortin & Boime, 1983;Wilson & Hatfield, 1984!. It has seen somewhat of a revival in recent years for recent examples, see Muller et al, 1994;Randhawa et al, 1994;Thomson et al, 1994;Budisa et al, 1995Budisa et al, , 1997Budisa et al, , 1998Xiao et al, 1997;Furter, 1998;Parsons et al, 1998!. When the unnatural amino acid is incorporated to a high level into the protein, one can potentially examine its effects on the protein's properties.…”

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confidence: 99%

Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: Biochemical and spectroscopic studies

Yuan

Vogel

1999

Protein Science

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Calmodulin~CaM! is a 148-residue regulatory calcium-binding protein that activates a wide range of target proteins and enzymes. Calcium-saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two "active sites" of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted~up to 90% incorporation! the unnatural amino acids ethionine~Eth! and norleucinẽ Nle! for the nine Met residues of CaM. The substituted proteins bind in a calcium-dependent manner to hydrophobic matrices and a synthetic peptide, encompassing the CaM-binding domain of myosin light-chain kinase~MLCK!. Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins compared to wild-type CaM~WT-CaM!. One-and two-dimensional NMR studies of the Eth-CaM and Nle-CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation studies with MLCK show that Eth-CaM and Nle-CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the K D for Nle-CaM also increased 3.5-fold compared with WT-CaM. These data show that the "active site" Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site-directed mutagenesis studies of the Met residues of CaM.Keywords: biosynthetic incorporation; calmodulin; ethionine; methionine; norleucine; spectroscopy; unnatural amino acid Calmodulin~CaM! is a ubiquitous calcium-regulatory protein that can bind and activate more than 40 different target proteins in eukaryotic cells~for recent reviews, see

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“…Conversely, the in vivo approach requires auxotrophic expression hosts. This method is much simpler, and incorporation works under the strong selective pressure (selective pressure incorporation) obtained by a stable auxotrophism and a controllable strong expression system, which leads to a residuespecific labeled protein (2,3).…”

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confidence: 99%

Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: Structure and stability of the per -thiaproline mutant of annexin V

Budiša

Minks

Medrano

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: Structure and stability of the per-thiaproline mutant of annexin V ABSTRACTResidue-specific bioincorporation of 1,3-thiazolidine-4-carboxylic acid [thiaproline, Pro(S)], a nonnatural amino acid analog of proline, into human recombinant annexin V was achieved with a proline-auxotrophic Escherichia coli strain by fermentation procedures in minimal medium. Quantitative replacement of proline with thiaproline was confirmed by mass-spectrometric, amino acid, and x-ray crystallographic analyses. The wild-type protein and its perPro(S) mutant were found to crystallize isomorphously and to show identical three-dimensional structures in crystals. In solution the dichroic properties of the wild-type and perPro(S) protein confirmed nearly identical overall folds. From thermal denaturation experiments, however, a reduced T m (؊4.5 K) value was determined whereas the van't Hoff enthalpy and entropy were not significantly affected. Therefore, protein mutants containing bioactive amino acid analogs like thiaproline at multiple sites would be expected to fully retain their functional properties, including immunogenicity, and thus could serve as promising vehicles for targeted drug delivery.

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Bioincorporation of telluromethionine into proteins: a promising new approach for X-ray structure analysis of proteins 1 1Edited by K. Nagai

Cited by 74 publications

References 17 publications

Selenium incorporation using recombinant techniques

Selenium incorporation using recombinant techniques

Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: Biochemical and spectroscopic studies

Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: Structure and stability of the per -thiaproline mutant of annexin V

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