Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG

2001 ◽  
Vol 353 (2) ◽  
pp. 395-401 ◽  
Author(s):  
Jennifer A. BECKINGHAM ◽  
Nicholas G. HOUSDEN ◽  
Nicola M. MUIR ◽  
Stephen P. BOTTOMLEY ◽  
Michael G. GORE
Keyword(s):  
Chain Complex ◽  
Binding Domain ◽  
Protein L ◽  
Human Igg ◽  
Tyrosine Residues ◽  
Kd Values ◽  
Trp Mutant ◽  
The Stability ◽  
Immunoglobulin Binding

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P. magnus strain 3316) and the kappa light chain (κ-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr51 and Tyr53) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr53. Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG. Modification of κ-chain by TNM resulted in the nitration of 3.1±0.09 tyrosine residues. When the PpLŐκ-chain complex was incubated with TNM, 4.1±0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The Kd for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112±20nM. A similar value (153±33nM) was obtained for the complex formed between IgG and the Tyr64 → Trp mutant (Y64W). However, the Kd values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2±0.2 and 4.6±1µM respectively. These suggest that the phenol group of Tyr53 in PpL is important to the stability of the PpLŐκ-chain complex.

scholarly journals Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG

2001 ◽  
Vol 353 (2) ◽  
pp. 395 ◽  
Author(s):  
Jennifer A. BECKINGHAM ◽  
Nicholas G. HOUSDEN ◽  
Nicola M. MUIR ◽  
Stephen P. BOTTOMLEY ◽  
Michael G. GORE
Keyword(s):  
Binding Domain ◽  
Protein L ◽  
Human Igg ◽  
Peptostreptococcus Magnus ◽  
Immunoglobulin Binding

The role of tyrosine residues of protein L in the binding reaction with human IgG

2001 ◽  
Vol 29 (1) ◽  
pp. A22-A22
Author(s):  
N. G. Housden ◽  
J. A. Beckingham ◽  
N. M. Muir ◽  
S. P. Bottomley ◽  
M. G. Gore
Keyword(s):  
Protein L ◽  
Human Igg ◽  
Binding Reaction ◽  
Tyrosine Residues

scholarly journals Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q

1989 ◽  
Vol 257 (3) ◽  
pp. 845-851 ◽  
Author(s):  
M N McCall ◽  
S B Easterbrook-Smith
Keyword(s):  
Receptor Site ◽  
Binding Activity ◽  
Human Igg ◽  
Tryptic Peptides ◽  
Tyrosine Residues ◽  
Rabbit Igg

Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338.

scholarly journals Interactions between a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus and a human κ light chain

1999 ◽  
Vol 340 (1) ◽  
pp. 193 ◽  
Author(s):  
Jennifer A. BECKINGHAM ◽  
Stephen P. BOTTOMLEY ◽  
Roger HINTON ◽  
Brian J. SUTTON ◽  
Michael G. GORE
Keyword(s):  
Light Chain ◽  
Binding Domain ◽  
Protein L ◽  
Peptostreptococcus Magnus ◽  
Immunoglobulin Binding

scholarly journals Interactions between a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus and a human κ light chain

1999 ◽  
Vol 340 (1) ◽  
pp. 193-199 ◽  
Author(s):  
Jennifer A. BECKINGHAM ◽  
Stephen P. BOTTOMLEY ◽  
Roger HINTON ◽  
Brian J. SUTTON ◽  
Michael G. GORE
Keyword(s):  
Conformational Transition ◽  
Bimolecular Reaction ◽  
Binding Domain ◽  
Tryptophan Residue ◽  
Stopped Flow ◽  
Wild Type ◽  
Protein L ◽  
Peptostreptococcus Magnus ◽  
Cd Studies ◽  
Immunoglobulin Binding

The placement of a tryptophan residue into a single Ig-binding-domain of protein L from Peptostreptococcus magnus has been used to examine the binding interactions between the binding domain and ĸ light chains (ĸ-chains). The fluorescence intensity of the mutant domain increases on the formation of a complex with ĸ-chains. This has been used to determine the Kd of the complex under a range of conditions by using both pre-equilibrium and equilibrium methods. The Kd values determined for the complex with ĸ-chains at a number of different pH values are very close to those obtained with the wild-type domain, indicating that the mutation has not substantially affected its binding properties. Examination of the reaction between the mutant domain and ĸ-chains by stopped-flow fluorescence shows that complex formation takes place by two discrete, sequential processes. A fast bimolecular reaction, with a rate constant of 8.3×105 M-1·s-1 (at pH 8.0 and 25 °C), is followed by a slow unimolecular process with a rate (1.45 s-1) that is independent of the concentration of the reactants. This suggests that a conformational change occurs after the initial encounter complex is formed. The dissociation of the complex at equilibrium occurs in a single process of rate 0.095 s-1 at pH 8.0 and 25 °C. Stopped-flow CD studies show that a slow decrease in ellipticity at 275 nm occurs with a rate of 1.3 s-1 when wild-type protein binds to ĸ-chains, suggesting that the conformational transition might involve a change in environment around one or more tyrosine residues.

scholarly journals A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

2020 ◽  
Author(s):  
Jose M. González de Cózar ◽  
Maria Carretero-Junquera ◽  
Grzegorz L. Ciesielski ◽  
Sini M. Miettinen ◽  
Markku Varjosalo ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Shotgun Proteomics ◽  
Binding Domain ◽  
The Novel ◽  
Binding Model ◽  
Protein Partners ◽  
The Stability ◽  
Protein Variants

ABSTRACTIn eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, which in Drosophila melanogaster (Dm) is exceptionally long, 115 amino acids. Molecular modeling predicted this extended linker to fold into a structure similar to the conserved HBD. We measured catalytic activity and substrate binding by EMSA and biolayer interferometry, using a deletion series of protein variants. Both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, whilst loss of the novel HBD led to a ∼90% drop in K[cat] with a decreased K[M], and a large increase in the stability of the RNA/DNA hybrid-enzyme complex. The findings support a bipartite binding model for the enzyme, whereby the second HBD facilitates dissociation of the active site from the product, allowing for processivity. We used shotgun proteomics to identify protein partners of the enzyme involved in mediating these effects. Single-stranded DNA-binding proteins from both the nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, were prominently detected by this method. However, we were not able to document direct interactions between mtSSB and Dm RNase H1 when co-overexpressed in S2 cells, or functional interactions in vitro. Further studies are needed to determine the exact reaction mechanism of Dm RNase H1, the nature of its interaction with mtSSB and the role of the second HBD in both.

scholarly journals Observation and Characterization of the Interaction between a Single Immunoglobulin Binding Domain of Protein L and Two Equivalents of Human κ Light Chains

2003 ◽  
Vol 279 (10) ◽  
pp. 9370-9378 ◽  
Author(s):  
Nicholas G. Housden ◽  
Steven Harrison ◽  
Hazel R. Housden ◽  
Karen-Anne Thomas ◽  
Jennifer A. Beckingham ◽  
...  
Keyword(s):  
Binding Domain ◽  
Light Chains ◽  
Protein L ◽  
Immunoglobulin Binding

A Statistical Study of Process Variables to Optimize a High Speed Curtain Coater: Part I

TAPPI Journal ◽  
2009 ◽  
Vol 8 (1) ◽  
pp. 20-26 ◽  
Author(s):  
PEEYUSH TRIPATHI ◽  
MARGARET JOYCE ◽  
PAUL D. FLEMING ◽  
MASAHIRO SUGIHARA
Keyword(s):  
Boundary Layer ◽  
Experimental Design ◽  
High Speed ◽  
Statistical Study ◽  
Process Variables ◽  
High Speeds ◽  
The Stability ◽  
Air Removal ◽  
Removal System

Using an experimental design approach, researchers altered process parameters and material prop-erties to stabilize the curtain of a pilot curtain coater at high speeds. Part I of this paper identifies the four significant variables that influence curtain stability. The boundary layer air removal system was critical to the stability of the curtain and base sheet roughness was found to be very important. A shear thinning coating rheology and higher curtain heights improved the curtain stability at high speeds. The sizing of the base sheet affected coverage and cur-tain stability because of its effect on base sheet wettability. The role of surfactant was inconclusive. Part II of this paper will report on further optimization of curtain stability with these four variables using a D-optimal partial-facto-rial design.

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