scholarly journals A New Practice to Monitor the Fabrication Process of Fab-Targeting Ligands from Bevacizumab by LC-MS: Preparation and Analytical Characterization

2022 ◽  
Vol 90 (1) ◽  
pp. 5
Author(s):  
Franck Marquet ◽  
Valentina D’Atri ◽  
Davy Guillarme ◽  
Gerrit Borchard
Keyword(s):  
Binding Sites ◽  
Disulfide Bonds ◽  
Digestive Enzyme ◽  
Reduction Reaction ◽  
Disulfide Bridges ◽  
Analytical Strategy ◽  
Purification Technique ◽  
The Reformation ◽  
Mild Reduction ◽  
Additional Processing

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as a model. The evaluation focused on the purification of fragments through a nonaffinity-based process using a centrifugal ultrafiltration technique and mild reduction conditions for the intact production of F(ab’) fragments with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, F(ab’) fragments with a defined chemical composition were successfully obtained via proteolytic digestion followed by a controlled reduction reaction process maintaining the integrity of the binding sites. The ultrafiltration purification technique appears to be suitable for the removal of the digestive enzyme but inefficient for the removal of Fc fragments, thus requiring additional processing. A suitable analytical strategy was developed, allowing us to demonstrate the reformation of disulfide bridges between the two reduced cysteines within F(ab’) fragments.

scholarly journals Stabilization of phage T4 lysozyme by engineered disulfide bonds

1989 ◽  
Vol 86 (17) ◽  
pp. 6562-6566 ◽  
Author(s):  
M Matsumura ◽  
W J Becktel ◽  
M Levitt ◽  
B W Matthews
Keyword(s):  
Disulfide Bonds ◽  
Theoretical Calculations ◽  
Equilibrium Constants ◽  
T4 Lysozyme ◽  
Disulfide Bridges ◽  
Large Loop ◽  
Phage T4 ◽  
Flexible Part ◽  
Free Phage

Four different disulfide bridges (linking positions 9-164, 21-142, 90-122, and 127-154) were introduced into a cysteine-free phage T4 lysozyme at sites suggested by theoretical calculations and computer modeling. The new cysteines spontaneously formed disulfide bonds on exposure to air in vitro. In all cases the oxidized (crosslinked) lysozyme was more stable than the corresponding reduced (noncrosslinked) enzyme toward thermal denaturation. Relative to wild-type lysozyme, the melting temperatures of the 9-164 and 21-142 disulfide mutants were increased by 6.4 degrees C and 11.0 degrees C, whereas the other two mutants were either less stable or equally stable. Measurement of the equilibrium constants for the reduction of the engineered disulfide bonds by dithiothreitol indicates that the less thermostable mutants tend to have a less favorable crosslink in the native structure. The two disulfide bridges that are most effective in increasing the stability of T4 lysozyme have, in common, a large loop size and a location that includes a flexible part of the molecule. The results suggest that stabilization due to the effect of the crosslink on the entropy of the unfolded polypeptide is offset by the strain energy associated with formation of the disulfide bond in the folded protein. The design of disulfide bridges is discussed in terms of protein flexibility.

scholarly journals Elucidating Binding Sites and Affinities of ERα Agonist and Antagonist to Human Alpha-Fetoprotein by in silico Modeling and Point Mutagenesis

2019 ◽  
Author(s):  
Nurbubu T. Moldogazieva ◽  
Daria S. Ostroverkhova ◽  
Nikolai N. Kuzmich ◽  
Vladimir V. Kadochnikov ◽  
Alexander A. Terentiev ◽  
...  
Keyword(s):  
Binding Sites ◽  
In Silico ◽  
Disulfide Bonds ◽  
Electrostatic Interactions ◽  
Molecular Mechanisms ◽  
3D Structure ◽  
Scoring Function ◽  
Alpha Fetoprotein ◽  
Ligand Interaction ◽  
Ligand Interactions

Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting variety of hydrophobic ligands including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth and this can be attributed to its estrogen-binding ability. Despite AFP has long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFP-ligand interaction remain obscure. In our study we constructed homology-based 3D model of human AFP (HAFP) with the purpose to perform docking of ERα ligands, three agonists (17β-estradiol, estrone and diethylstilbestrol) and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on ligand docked scoring function, we identified three putative estrogen- and antiestrogen-binding sites with different ligand binding affinities. Two high-affinity sites were located in (i) a tunnel formed within HAFP subdomains IB and IIA and (ii) opposite side of the molecule in a groove originating from cavity formed between domains I and III, while (iii) the third low-affinity site was found at the bottom of the cavity. 100 ns MD simulation allowed studying their geometries and showed that HAFP-estrogen interactions occur due to van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP-antiestrogen binding. MM/GBSA rescoring method estimated binding free energies (ΔGbind) and showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP-ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues along two disulfide bonds, Cys224-Cys270 and Cys269-Cys277 have key roles in both HAFP-estrogen and HAFP-antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein-ligand interactions and anti-cancer therapy strategies based on ER-binding ligands.

scholarly journals Sindbis Virus Glycoprotein E1 Is Divided into Two Discrete Domains at Amino Acid 129 by Disulfide Bridge Connections

2000 ◽  
Vol 74 (19) ◽  
pp. 9313-9316 ◽  
Author(s):  
Brett S. Phinney ◽  
Dennis T. Brown
Keyword(s):  
Amino Acid ◽  
Disulfide Bonds ◽  
Sindbis Virus ◽  
Disulfide Bridge ◽  
Membrane Glycoprotein ◽  
Disulfide Bridges ◽  
Functional Regions ◽  
E1 Protein ◽  
Disulfide Linkages ◽  
Discrete Domains

ABSTRACT The E1 membrane glycoprotein of Sindbis virus contains structural and functional domains, which are conformationally dependent on the presence of intramolecular disulfide bridges (B. A. Abell and D. T. Brown, J. Virol. 67:5496–5501, 1993; R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330–336, 1992). We have examined the disulfide bonds in E1 and have determined that the E1 membrane glycoprotein contains two separate sets of interconnecting disulfide linkages, which divide the protein into two domains at amino acid 129. These separate sets of disulfides may stabilize and define the structural and functional regions of the E1 protein.

scholarly journals MetalDetector: a web server for predicting metal-binding sites and disulfide bridges in proteins from sequence

2008 ◽  
Vol 24 (18) ◽  
pp. 2094-2095 ◽  
Author(s):  
M. Lippi ◽  
A. Passerini ◽  
M. Punta ◽  
B. Rost ◽  
P. Frasconi
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Web Server ◽  
Disulfide Bridges ◽  
Metal Binding Sites

scholarly journals Isolation of insoluble secretory product from bovine thyroid: extracellular storage of thyroglobulin in covalently cross-linked form.

1992 ◽  
Vol 118 (5) ◽  
pp. 1071-1083 ◽  
Author(s):  
V Herzog ◽  
U Berndorfer ◽  
Y Saber
Keyword(s):  
Disulfide Bonds ◽  
Plasma Membranes ◽  
Matrix Protein ◽  
Thyroid Tissue ◽  
Surface Pattern ◽  
Secretory Product ◽  
Disulfide Bridges ◽  
Luminal Content ◽  
Bovine Thyroid ◽  
A Minor

Extracellular storage of thyroglobulin (TG) is an important prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of large amounts is made possible by compactation of TG in the follicle lumen with concentrations of at least 100-400 mg/ml. We recently observed that the luminal content from bovine thyroids can be isolated in an intact state and be separated from soluble TG. For this purpose, bovine thyroid tissue was homogenized and subjected to various steps of purification. This procedure resulted in a pellet of single globules measuring 20-120 microns in diameter. Scanning electron microscopy revealed a unique cobblestone-like surface pattern of isolated globules, showing in detail the impressions of the apical plasma membranes of thyrocytes which had formerly surrounded the luminal content before tissue homogenization. Isolated thyroid globules were rapidly digested by trypsin but extremely resistant to various protein solubilization procedures. Homogenization of isolated globules resulted in the release of approximately 3% of total protein, showing that only a minor proportion of TG was loosely incorporated in thyroid globules whereas approximately 22% appeared to be interconnected with the globule matrix by disulfide bridges. Analysis by SDS-gel electrophoresis and immunoblotting confirmed that the protein released by this procedure consisted of TG. The vast majority (approximately 75%) of the globule matrix protein was found to be covalently cross-linked by non-disulfide bonds. TG in isolated globules was highly iodinated (approximately 55 iodine atoms per 12-S TG subunit) suggesting that the covalent nondisulfide cross-linking occurs in part during the iodination of TG and that this process involves the formation of intermolecular dityrosine bridges. Mechanisms must exist which solubilize or disperse the insoluble luminal content prior to endocytosis of TG.

scholarly journals Identification of Iron-Sulfur (Fe-S) and Zn-binding Sites Within Proteomes Predicted by DeepMind′s AlphaFold2 Program Dramatically Expands the Metalloproteome

2021 ◽  
Author(s):  
Zachary J Wehrspan ◽  
Robert T McDonnell ◽  
Adrian Elcock
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Structure Prediction ◽  
Disulfide Bonds ◽  
3D Protein Structure ◽  
E Coli ◽  
Iron Sulfur ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Binding Orientations

DeepMind′s AlphaFold2 software has ushered in a revolution in high quality, 3D protein structure prediction. In very recent work by the DeepMind team, structure predictions have been made for entire proteomes of twenty-one organisms, with >360,000 structures made available for download. Here we show that thousands of novel binding sites for iron-sulfur (Fe-S) clusters and zinc ions can be identified within these predicted structures by exhaustive enumeration of all potential ligand-binding orientations. We demonstrate that AlphaFold2 routinely makes highly specific predictions of ligand binding sites: for example, binding sites that are comprised exclusively of four cysteine sidechains fall into three clusters, representing binding sites for 4Fe-4S clusters, 2Fe-2S clusters, or individual Zn ions. We show further: (a) that the majority of known Fe-S cluster and Zn-binding sites documented in UniProt are recovered by the AlphaFold2 structures, (b) that there are occasional disputes between AlphaFold2 and UniProt with AlphaFold2 predicting highly plausible alternative binding sites, (c) that the Fe-S cluster binding sites that we identify in E. coli agree well with previous bioinformatics predictions, (d) that cysteines predicted here to be part of Fe-S cluster or Zn-binding sites show little overlap with those shown via chemoproteomics techniques to be highly reactive, and (e) that AlphaFold2 occasionally appears to build erroneous disulfide bonds between cysteines that should instead coordinate a ligand. These results suggest that AlphaFold2 could be an important tool for the functional annotation of proteomes, and the methodology presented here is likely to be useful for predicting other ligand-binding sites.

scholarly journals Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction

2021 ◽  
Vol 22 (20) ◽  
pp. 11142
Author(s):  
Yun-Ru Chen ◽  
Nai-Wan Hsiao ◽  
Yi-Zong Lee ◽  
Shiau-Shan Huang ◽  
Chih-Chun Chang ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Receptor Binding ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Receptor Interaction ◽  
Resonance Spectroscopy ◽  
Molecular Architecture ◽  
Major Factors

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; and α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7–Cys43, Cys23–Cys39, and Cys26–Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand–receptor binding specificity.

scholarly journals Role of Glutaredoxin-1 and Glutathionylation in Cardiovascular Diseases

2020 ◽  
Vol 21 (18) ◽  
pp. 6803 ◽  
Author(s):  
Mannix Burns ◽  
Syed Husain Mustafa Rizvi ◽  
Yuko Tsukahara ◽  
David R. Pimentel ◽  
Ivan Luptak ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Disulfide Bonds ◽  
Arterial Disease ◽  
Disulfide Bridges ◽  
Myocardial Ischemia And Reperfusion ◽  
Cellular Processes ◽  
Mixed Disulfide ◽  
Peripheral Arterial ◽  
Structure Of Proteins

Cardiovascular diseases are the leading cause of death worldwide, and as rates continue to increase, discovering mechanisms and therapeutic targets become increasingly important. An underlying cause of most cardiovascular diseases is believed to be excess reactive oxygen or nitrogen species. Glutathione, the most abundant cellular antioxidant, plays an important role in the body’s reaction to oxidative stress by forming reversible disulfide bridges with a variety of proteins, termed glutathionylation (GSylation). GSylation can alter the activity, function, and structure of proteins, making it a major regulator of cellular processes. Glutathione-protein mixed disulfide bonds are regulated by glutaredoxins (Glrxs), thioltransferase members of the thioredoxin family. Glrxs reduce GSylated proteins and make them available for another redox signaling cycle. Glrxs and GSylation play an important role in cardiovascular diseases, such as myocardial ischemia and reperfusion, cardiac hypertrophy, peripheral arterial disease, and atherosclerosis. This review primarily concerns the role of GSylation and Glrxs, particularly glutaredoxin-1 (Glrx), in cardiovascular diseases and the potential of Glrx as therapeutic agents.

Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Ana Victoria Bendetowicz ◽  
Jill A. Morris ◽  
Robert J. Wise ◽  
Gary E. Gilbert ◽  
Randal J. Kaufman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Free System ◽  
N Terminus ◽  
A Cell ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

Vasodilation and glomerular binding of adrenomedullin in rabbit kidney are not CGRP receptor mediated

1997 ◽  
Vol 273 (2) ◽  
pp. R716-R724 ◽  
Author(s):  
H. Hjelmqvist ◽  
R. Keil ◽  
M. Mathai ◽  
T. Hubschle ◽  
R. Gerstberger
Keyword(s):  
Binding Sites ◽  
Membrane Receptors ◽  
Rabbit Kidney ◽  
Calcitonin Gene ◽  
Body Fluid Homeostasis ◽  
Mild Reduction ◽  
Specific Membrane ◽  
Camp Formation

The polypeptide adrenomedullin (ADM) was infused systemically to conscious rabbits to elucidate its actions on overall circulation and especially the renovascular bed and the formation and/or release of hormones important for body fluid homeostasis, including adrenocortical steroids. ADM lowered mean arterial pressure from 71.5 +/- 3.2 to 64.7 +/- 3.2 mmHg only at the highest dose of 25 pmol.min-1.kg-1 infused intravenously for 20 min and concomitantly induced tachycardia, possibly due to both baroreflex activation and direct cardiostimulatory effects. Renal blood flow (RBF) determined in rabbits chronically equipped with a perivascular ultrasonic flow probe increased from 55.4 +/- 2.1 to 67.4 +/- 2.7 and from 58.2 +/- 3.5 to 75.2 +/- 6.0 ml/min at ADM infusions of 5 and 25 pmol.min-1.kg-1, respectively. The elevation in RBF persisted even in the presence of the calcitonin gene-related peptide (CGRP1 receptor antagonist CGRP-(8-37). Of all osmoregulatory hormones tested, only corticosterone (Cort) plasma concentration increased in response to the highest ADM dose from 17.6 +/- 3.1 to 38.9 +/- 6.2 ng/ml, probably due to haroreflex activation. Subdepressor doses of ADM, however, caused a mild reduction in circulating Cort. Expression of functional high-affinity binding sites specific for ADM in vitro could be demonstrated for the renal artery and outer cortical glomeruli using 125I-labeled rat ADM as radioligand and determination of cellular adenosine 3',5'-cyclic monophosphate (cAMP) formation within the glomeruli. The ineffectiveness of CGRP-(8-37) to displace radiolabeled ADM from its binding sites, to inhibit ADM-induced glomerular cAMP formation, and to prevent ADM-induced renal vasodilation supports the hypothesis of ADM altering renal hemodynamics by interacting with ADM- and not CGRP-specific membrane receptors.

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