Molecular Recognition in the P450cam Monooxygenase System: Direct Monitoring of Protein–Protein Interactions by Using Optical Biosensor

2001 ◽  
Vol 391 (2) ◽  
pp. 255-264 ◽  
Author(s):  
Yuri D. Ivanov ◽  
Irina P. Kanaeva ◽  
Irina I. Karuzina ◽  
Alexander I. Archakov ◽  
Gaston Hui Bon Hoa ◽  
...  
Keyword(s):  
Molecular Recognition ◽  
Protein Interactions ◽  
Optical Biosensor ◽  
Monooxygenase System ◽  
Protein Protein Interactions

scholarly journals Detection of Protein-Protein Interactions in the Alkanesulfonate Monooxygenase System from Escherichia coli

2006 ◽  
Vol 188 (23) ◽  
pp. 8153-8159 ◽  
Author(s):  
Kholis Abdurachim ◽  
Holly R. Ellis
Keyword(s):  
Circular Dichroism ◽  
Protein Interactions ◽  
Visible Region ◽  
Circular Dichroism Spectroscopy ◽  
Flavin Mononucleotide ◽  
Stable Complex ◽  
Monooxygenase System ◽  
Protein Protein Interactions ◽  
Alkanesulfonate Monooxygenase ◽  
Circular Dichroïsm

ABSTRACT The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The importance of protein-protein interactions in the mechanism of flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD). Interactions between the two proteins do not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.0022 ± 0.0010 μM was obtained for the binding of SsuE to SsuD. Based on these studies, the stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD.

Protein-Protein Interactions in the P450 Monooxygenase System

1999 ◽  
pp. 21-39
Author(s):  
John B. Schenkman ◽  
Ingela Jansson ◽  
Gary Davis ◽  
Paul P. Tamburini ◽  
Zhongqing Lu ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Monooxygenase System ◽  
Protein Protein Interactions ◽  
P450 Monooxygenase

Kinetics of Protein-Protein Interactions at the Surface of an Optical Biosensor

1995 ◽  
Vol 231 (1) ◽  
pp. 210-217 ◽  
Author(s):  
P.R. Edwards ◽  
A. Gill ◽  
D.V. Pollardknight ◽  
M. Hoare ◽  
P.E. Buckle ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Optical Biosensor ◽  
Protein Protein Interactions ◽  
Kinetics Of

scholarly journals Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5

2015 ◽  
Vol 61 (4) ◽  
pp. 468-474
Author(s):  
O.V. Gnedenko ◽  
A.S. Ivanov ◽  
E.O. Yablokov ◽  
S.A. Usanov ◽  
D.V. Mukha ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Dissociation Constants ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Electrochemical Analysis ◽  
Monooxygenase System ◽  
Protein Protein Interactions ◽  
Reduction Potentials ◽  
Drug Biotransformation ◽  
Redox Partners

Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3A4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 “chimeric” proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes

2014 ◽  
Vol 60 (1) ◽  
pp. 17-27 ◽  
Author(s):  
O.V. Gnedenko ◽  
A.S. Ivanov ◽  
E.O. Yablokov ◽  
S.A. Usanov ◽  
D.V. Mukha ◽  
...  
Keyword(s):  
Cytochrome B ◽  
Protein Interactions ◽  
Dissociation Constants ◽  
Cytochromes P450 ◽  
Electrochemical Analysis ◽  
Monooxygenase System ◽  
Protein Protein Interactions ◽  
Reduction Potentials ◽  
Drug Biotransformation ◽  
Redox Partners

Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b ) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b : the microsomal ( b5mc ) and mitochondrial ( b5om ) forms of this protein, as well as with 2 “chimeric” proteins, b5(om-mc) , b5(mc-om) . Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om . Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om , b5(om-mc) , and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435- -0.350 V (vs. Ag/AgCl). Cytochrome b mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 to testosterone.

scholarly journals Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders

2017 ◽  
Vol 61 (5) ◽  
pp. 505-516 ◽  
Author(s):  
Scott J. Hughes ◽  
Alessio Ciulli
Keyword(s):  
Molecular Recognition ◽  
Drug Design ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Structural Data ◽  
Ternary Complexes ◽  
Protein Protein Interactions ◽  
New Paradigm ◽  
Design And Optimization ◽  
Three Body

Molecular glues and bivalent inducers of protein degradation (also known as PROTACs) represent a fascinating new modality in pharmacotherapeutics: the potential to knockdown previously thought ‘undruggable’ targets at sub-stoichiometric concentrations in ways not possible using conventional inhibitors. Mounting evidence suggests these chemical agents, in concert with their target proteins, can be modelled as three-body binding equilibria that can exhibit significant cooperativity as a result of specific ligand-induced molecular recognition. Despite this, many existing drug design and optimization regimens still fixate on binary target engagement, in part due to limited structural data on ternary complexes. Recent crystal structures of protein complexes mediated by degrader molecules, including the first PROTAC ternary complex, underscore the importance of protein–protein interactions and intramolecular contacts to the mode of action of this class of compounds. These discoveries have opened the door to a new paradigm for structure-guided drug design: borrowing surface area and molecular recognition from nature to elicit cellular signalling.

scholarly journals The effect of isatin on protein-protein interactions between cytochrome b5 and cytochromes P450

2017 ◽  
Vol 63 (2) ◽  
pp. 170-175 ◽  
Author(s):  
P.V. Ershov ◽  
E.O. Yablokov ◽  
Yu.V. Mezentsev ◽  
L.A. Kalushskiy ◽  
A.V. Florinskaya ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Contact Region ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Optical Biosensor ◽  
Test System ◽  
Feedback Mechanism ◽  
Dissociation Rate ◽  
Protein Protein Interactions ◽  
High Concentration

Cytochromes P450 (CYP) are involved in numerous biochemical processes including metabolism of xenobiotics, biosynthesis of cholesterol, steroid hormones etc. Since some CYP catalyze indol oxidation to isatin, we have hypothesized that isatin can regulate protein-protein interactions (PPI) between components of the CYP system thus representing a (negative?) feedback mechanism. The aim of this study was to investigate a possible effect of isatin on interaction of human CYP with cytochrome b5 (CYB5A). Using the optical biosensor test system employing surface plasmon resonance (SPR) we have investigated interaction of immobilized CYB5A with various CYP in the absence and in the presence of isatin. The SPR-based experiments have shown that a high concentration of isatin (270 mM) increases Kd values for complexes CYB5A/CYP3А5 and CYB5A/CYP3A4 (twofold and threefold, respectively), but has no influence on complex formation between CYB5A and other CYP (including indol-metabolizing CYP2C19 and CYP2E1). Isatin injection to the optical biosensor chip with the preformed molecular complex CYB5A/CYP3A4 caused a 30%-increase in its dissociation rate. Molecular docking manipulations have shown that isatin can influence interaction of CYP3А5 or CYP3A4 with CYB5A acting at the contact region of CYB5A/CYP.

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