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

Budiša, Nediljko; Minks, Caroline; Medrano, Francisco J.; Lutz, Jürgen; Huber, Robert; Moröder, Luis

doi:10.1073/pnas.95.2.455

Cited by 89 publications

(92 citation statements)

References 19 publications

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“…Proline Analogs Affect Full-length Tagging-The proline analogs azetidine-2-carboxylic acid, ␥-thiaproline, and 3,4-dehydroproline are incorporated into proteins in E. coli (26,27). Using a proline auxotrophic strain, we examined the effects of these proline analogs on SsrA tagging of a variant of YbeL containing a His 6 sequence before the last four residues of the wild-type protein (Fig.…”

Section: Altered Levels Of Translation Termination Factors Affectmentioning

confidence: 99%

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

Hayes¹,

Bose

Sauer

2002

Journal of Biological Chemistry

145

177

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The SsrA or tmRNA quality control system relieves ribosome stalling and directs the addition of a degradation tag to the C terminus of the nascent chain. In some instances, SsrA tagging of otherwise full-length proteins occurs when the ribosome pauses at stop codons during normal translation termination. Here, the identities of the C-terminal residues of the nascent chain are shown to play an important role in full-length protein tagging. Specifically, a subset of C-terminal Xaa-Pro sequences caused SsrA tagging of the full-length YbeL protein from Escherichia coli. This tagging increased when a less efficient stop codon was used, increased in cells lacking protein release factor-3, and decreased when protein release factor-1 was overexpressed. Incorporation of the analog azetidine-2-carboxylic acid in place of proline suppressed tagging, whereas incorporation of 3,4-dehydroproline increased SsrA tagging of full-length YbeL. These results suggest that the detailed chemical or conformational properties of the C-terminal residues of the nascent polypeptide can affect the rate of translation termination, thereby influencing ribosome pausing and SsrA tagging at stop codons.

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Section: Altered Levels Of Translation Termination Factors Affectmentioning

confidence: 99%

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

Hayes¹,

Bose

Sauer

2002

Journal of Biological Chemistry

145

177

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“…T4C Survival Assay-T4C competes with Pro in the translation process (42,43) and can be degraded by Pro dehydrogenase in plants and bacteria (44,45). One-week-old WT and ProDH-OE tobacco seedlings (T 2 ) were transferred to MS medium containing 50 mM NaCl, 3 mM T4C, or 50 mM NaCl ϩ 3 mM T4C.…”

Section: Methodsmentioning

confidence: 99%

Unraveling Δ1-Pyrroline-5-Carboxylate-Proline Cycle in Plants by Uncoupled Expression of Proline Oxidation Enzymes

Miller

Honig

Stein

et al. 2009

Journal of Biological Chemistry

229

221

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The two-step oxidation of proline in all eukaryotes is performed at the inner mitochondrial membrane by the consecutive action of proline dehydrogenase (ProDH) that produces ⌬ 1 -pyrroline-5-carboxylate (P5C) and P5C dehydrogenase (P5CDH) that oxidizes P5C to glutamate. This catabolic route is down-regulated in plants during osmotic stress, allowing free Pro accumulation. We show here that overexpression of MsProDH in tobacco and Arabidopsis or impairment of P5C oxidation in the Arabidopsis p5cdh mutant did not change the cellular Pro to P5C ratio under ambient and osmotic stress conditions, indicating that P5C excess was reduced to Pro in a mitochondrial-cytosolic cycle. This cycle, involving ProDH and P5C reductase, exists in animal cells and now demonstrated in plants. As a part of the cycle, Pro oxidation by the ProDH-FAD complex delivers electrons to the electron transport chain. Hyperactivity of the cycle, e.g. when an excess of exogenous L-Pro is provided, generates mitochondrial reactive oxygen species (ROS) by delivering electrons to O 2 , as demonstrated by the mitochondria-specific MitoSox staining of superoxide ions. Lack of P5CDH activity led to higher ROS production under dark and light conditions in the presence of Pro excess, as well as rendered plants hypersensitive to heat stress. Balancing mitochondrial ROS production during increased Pro oxidation is therefore critical for avoiding Pro-related toxic effects. Hence, normal oxidation of P5C to Glu by P5CDH is key to prevent P5C-Pro intensive cycling and avoid ROS production from electron run-off.Stress-related changes in Pro biosynthesis and degradation are conserved in different organisms and used to induce essential signaling pathways, such as p53-mediated apoptosis in mammalian cells (1) or osmo-protecting mechanisms in plants (2) and bacteria (3). Pro is one of the most common osmolytes that, together with glycine-betaine and soluble sugars, accumulates in plants in response to a wide array of abiotic and biotic stresses (2,4,5). Although the role of Pro as an osmo-protecting molecule in prokaryotes is well established (3, 6), the overall function of stress-accumulated free Pro in plants is still under debate (2, 7-9). In most plants, stress-induced free Pro accumulation results from enhanced Pro synthesis and concomitant silencing of Pro degradation (10 -13). Pro is produced in the cytosol and chloroplasts (14, 15) and degraded in the mitochondria by a short, strictly regulated cyclic pathway (Fig. 1). In the anabolic part of the pathway, glutamate is converted to ⌬ 1 -pyrroline-5-carboxylate (P5C) 3 by P5C synthetase (P5CS), and P5C-reductase (P5CR) reduces P5C to Pro. In an alternative pathway, P5C is generated from ornithine by mitochondrial ornithine-aminotransferase (16, 17) and is reduced to Pro by P5CR in the cytosol. Thus, a possible movement of P5C among organelles and cytosol is assumed. P5CS is considered the key enzyme in the synthesis route, and its expression is increased by osmotic stress in many plants (11, 16 -18). A singl...

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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.…”

mentioning

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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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

Cited by 89 publications

References 19 publications

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

Unraveling Δ1-Pyrroline-5-Carboxylate-Proline Cycle in Plants by Uncoupled Expression of Proline Oxidation Enzymes

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

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