Escherichia coli
          l-aspartate-α-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry

The Escherichia coli panD gene, encoding l-aspartate-α-decarboxylase, was cloned by PCR, and shown to complement apanD mutant defective in β-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the π-protein), which is proteolytically cleaved at a specific X–Ser bond to produce a β-subunit with XOH at its C-terminus and an α-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed π-subunit (of 13.8 kDa), with a small proportion of the α- and β-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the α-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151±16 μM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.

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Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

Journal of Bacteriology ◽

10.1128/jb.00407-08 ◽

2008 ◽

Vol 190 (15) ◽

pp. 5517-5521 ◽

Cited By ~ 11

Author(s):

Edan R. Hosking ◽

Michael D. Manson

Keyword(s):

Escherichia Coli ◽

Flagellar Motor ◽

Protein Levels ◽

Partner Protein ◽

C Terminus ◽

N Terminus ◽

Charged Residues ◽

Positively Charged ◽

Terminal Cluster

ABSTRACT MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

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The N Terminus of GTPγS-activated Transducin α-Subunit Interacts with the C Terminus of the cGMP Phosphodiesterase γ-Subunit

Journal of Biological Chemistry ◽

10.1074/jbc.m509511200 ◽

2006 ◽

Vol 281 (10) ◽

pp. 6194-6202 ◽

Cited By ~ 17

Author(s):

Jennifer E. Grant ◽

Lian-Wang Guo ◽

Martha M. Vestling ◽

Kirill A. Martemyanov ◽

Vadim Y. Arshavsky ◽

...

Keyword(s):

Α Subunit ◽

Cgmp Phosphodiesterase ◽

Γ Subunit ◽

C Terminus ◽

N Terminus

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Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

Journal of Molecular Endocrinology ◽

10.1677/jme-07-0151 ◽

2008 ◽

Vol 40 (4) ◽

pp. 185-198 ◽

Cited By ~ 14

Author(s):

Sébastien Legardinier ◽

Jean-Claude Poirier ◽

Danièle Klett ◽

Yves Combarnous ◽

Claire Cahoreau

Keyword(s):

Thermal Stability ◽

Chorionic Gonadotropin ◽

Sf9 Cells ◽

Β Subunit ◽

Single Chain ◽

Α Subunit ◽

C Terminus ◽

N Terminus ◽

Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

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Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of metE and metH: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase

Journal of Bacteriology ◽

10.1128/jb.182.19.5539-5550.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5539-5550 ◽

Cited By ~ 15

Author(s):

Paula S. Fritsch ◽

Mark L. Urbanowski ◽

George V. Stauffer

Keyword(s):

Rna Polymerase ◽

Random Mutagenesis ◽

Complex Surface ◽

Α Subunit ◽

C Terminus ◽

Distinct Cluster ◽

In Vitro Reconstitution ◽

The Difference ◽

Α Subunits

ABSTRACT Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α2, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the αCTD on the β-associated α subunit at metH. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.

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Epitope characterization by modifications of antigens and by mapping on resin-bound peptides Discriminating epitopes near the C-terminus and N-terminus of Escherichia coli ribosomal protein S13

Journal of Immunological Methods ◽

10.1016/0022-1759(89)90001-x ◽

1989 ◽

Vol 118 (2) ◽

pp. 153-160 ◽

Cited By ~ 11

Author(s):

Wan-Jr Syu ◽

Lawrence Kahan

Keyword(s):

Escherichia Coli ◽

Ribosomal Protein ◽

C Terminus ◽

N Terminus ◽

Epitope Characterization ◽

Ribosomal Protein S13

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Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

Journal of Cell Science ◽

10.1242/jcs.99.4.823 ◽

1991 ◽

Vol 99 (4) ◽

pp. 823-836

Author(s):

S.J. Atkinson ◽

M. Stewart

Keyword(s):

Escherichia Coli ◽

Ionic Strength ◽

Coiled Coil ◽

Periodic Variation ◽

Heptad Repeat ◽

C Terminus ◽

N Terminus ◽

Low Ionic Strength ◽

Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

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Syntheses and stabilities of proteins related to the polyoma small T antigen in Escherichia coli

Molecular and Cellular Biology ◽

10.1128/mcb.2.1.88-92.1982 ◽

1982 ◽

Vol 2 (1) ◽

pp. 88-92

Author(s):

A Horwich ◽

A H Koop ◽

W Eckhart

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

T Antigen ◽

Bacterial Proteins ◽

N Terminus ◽

Terminal Amino ◽

Small T Antigen ◽

Beta Galactosidase

We compared the syntheses and turnovers of two proteins related to the polyoma small T antigen synthesized in Escherichia coli from plasmids containing polyoma genomic segments joined to lac control elements. A protein with an authentic polyoma N terminus was more unstable than a protein with N-terminal amino acids derived from beta-galactosidase. Both were more unstable than most bacterial proteins.

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Does Cysteine Rule (CysR) Complete the CendR Principle? Increase in Affinity of Peptide Ligands for NRP-1 Through the Presence of N-Terminal Cysteine

Biomolecules ◽

10.3390/biom10030448 ◽

2020 ◽

Vol 10 (3) ◽

pp. 448 ◽

Cited By ~ 1

Author(s):

Anna K. Puszko ◽

Piotr Sosnowski ◽

Françoise Raynaud ◽

Olivier Hermine ◽

Gérard Hopfgartner ◽

...

Keyword(s):

Peptide Ligands ◽

Side Chain ◽

Amino Group ◽

Structure Activity ◽

C Terminus ◽

Neuropilin 1 ◽

Ic50 Value ◽

N Terminus ◽

Terminal Amino ◽

Relationship Of

The structure-activity relationship of branched H-Lys(hArg)-Dab-Dhp-Arg-OH sequence analogues, modified with Cys-Asp or Cys at N-terminal amino acids (Lys, hArg), in VEGF-A165/Neuropilin-1 complex inhibition is presented. The addition of Cys residue led to a 100-fold decrease in the IC50 value, compared to the parent peptide. The change occurred regardless of coupling Cys to the free N-terminal amino group present in the main or the side chain. A few analogues extended by the attachment of Cys at the N-terminus of several potent NRP-1 peptide ligands documented in the literature are also presented. In all studied cases, the enhancement of inhibitory properties after the addition of Cys at the N-terminus is observed. It is particularly evident for the tetrapeptide derived from the C-terminus of VEGF-A165 (KPRR), suggesting that extending the K/RXXK/R motif (CendR) with the Cys moiety can significantly improve affinity to NRP-1 of CendR peptides.

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