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Serine Mutant (serine + mutant)

Distribution by Scientific Domains
Chemistry75%

Selected Abstracts

Nuclear factor-,B contributes to interleukin-4- and interferon-dependent polymeric immunoglobulin receptor expression in human intestinal epithelial cells

IMMUNOLOGY, Issue 1 2004
Laynez W. Ackermann
Summary Polymeric immunoglobulins (pIgs) that are present at mucosal surfaces play key roles in both the innate and adaptive immune responses. These pIgs are delivered to the mucosal surface via transcytosis across the epithelium, a process mediated by the polymeric immunoglobulin receptor (pIgR). Previous studies demonstrate that expression of the pIgR is regulated by multiple immunomodulatory factors including interleukin-4 (IL-4) and interferon-, (IFN-,). In studies using human intestinal epithelial cells (HT29), multiple inhibitors of the transcription factor nuclear factor-,B (NF-,B), including a dominant negative I,B,-serine mutant, inhibited both IL-4- and IFN-dependent increases in pIgR expression. Under identical conditions, NF-,B inhibitors had no effect on cytokine-dependent increases in expression of the transcription factor interferon regulatory factor-1. Over-expression of the I,B,-serine mutant also inhibited reporter gene expression in response to IL-4, TNF-,, IL-1,, and in some cases IFN-, using constructs with sequences from the pIgR promoter. Reduced levels of pIgR were observed even when inhibitors were added ,24 hr after cytokines suggesting that prolonged activation of NF-,B is required. Finally, reporter gene studies with NF-,B enhancer elements indicated that IFN-, alone and IL-4 in combination with other cytokines activated NF-,B in HT29 cells. Together, these studies provide additional insight into the signalling pathways that contribute to expression of the pIgR, a critical player in mucosal immunity. [source]

Expression, purification, crystallization and preliminary X-ray analysis of the native class C ,-­lactamase from Enterobacter cloacae 908R and two mutants

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2001
J. Wouters
Crystals have been obtained of the Enterobacter cloacae 908R ,-­lactamase and two point mutants by the vapour-diffusion method using similar conditions [pH 9.0, polyethylene glycol (Mr = 6000) as precipitant]. The three crystal forms belong to the orthorhombic space group P21212, with roughly the same unit-cell parameters; i.e. for the wild-type crystals a = 46.46, b = 82.96, c = 95.31,Å. In the best cases, the crystals diffract to about 2.1,Å resolution on a rotating-anode X-ray source at room temperature. Co-crystallization experiments of poor substrates with the wild-type protein and the active-site serine mutant (S64C) are planned and should lead to a better understanding of the catalytic mechanism of class C ,-lactamases. [source]

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferase 1 (GalNAc-T1)

FEBS JOURNAL, Issue 17 2002
Mari Tenno
Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p- chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc. [source]

New structural insights from Raman spectroscopy of proteins and their assemblies

BIOPOLYMERS, Issue 4-5 2002
George J. Thomas Jr.Article first published online: 9 MAY 200
Abstract Protein structure and stability are sensitive to and dependent on the local interactions of amino acid side chains. A diverse and important type of side-chain interaction is the hydrogen bond. Although numerous hydrogen bonds are resolved in protein 3-dimensional structures, those of the cysteine sulfhydryl group (S H) are elusive to high-resolution X-ray and NMR methods. However, the nature and strength of sulfhydryl hydrogen bonds (SH,X) are amenable to investigation by Raman spectroscopy. The power of the Raman method for characterizing SH,X interactions is illustrated by resolving the Raman SH stretching band for each of the eight cysteines per 666-residue subunit in the trimeric tailspike of icosahedral bacteriophage P22. The Raman sulfhydryl signatures of the wild-type tailspike and eight single-site cysteine to serine mutants reveal a heretofore unrecognized diversity of SH hydrogen bonds in a native protein. The use of Raman spectroscopy to identify the non-hydrogen-bonded state of the tyrosine phenoxyl group is also described. This unusual and unexpected state occurs for all tyrosines in the assembled capsids of filamentous viruses Ff and Pf1. The Raman spectral signature of the non-hydrogen-bonded tyrosine phenoxyl, which is characterized by an extraordinary Raman Fermi doublet intensity ratio (I850/I830 = 6.7), extends and refines the existing correlation for hydrogen-bonded tyrosines. Finally, a novel Raman signature for tryptophan in the Pf3 filamentous virus is identified, which is proposed as diagnostic of "cation,, interaction" involving the guanidinium group of Arg 37 as a cation donor and the indolyl ring of Trp 38 as a ,-electron acceptor. These studies demonstrate the power of Raman spectroscopy for investigating the interactions of key side chains in native protein assemblies. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 214,225, 2002 [source]