Computational Analysis of C-Reactive Protein for Assessment of Molecular Dynamics and Interaction Properties

Chakraborty, Chiranjib; Agrawal, Alok

doi:10.1007/s12013-013-9553-4

Cited by 10 publications

(7 citation statements)

References 83 publications

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“…Additionally, the increased electrophoretic migration of mCRP compared to pCRP (Fig. 3 ) is an experimental proof that salt-bridge electrostatic charges reported to stabilize the tightly compacted and associated pCRP (Thompson et al 1999 ; Agrawal and Chakraborty 2013 ) are broken when mCRP is expressed. When these intramolecular forces are relaxed, the free electrostatic charges now allow the protein to migrate faster (as an alpha globulin) contrasted to the reported gamma migration for pCRP (Laurent et al 1983 ).…”

Section: Discussionmentioning

confidence: 59%

Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification

Potempa

Yao

et al. 2015

Biophys. Rep.

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The precise function of C-reactive protein (CRP) as a regulator of inflammation in health and disease continues to evolve. The true understanding of its role in host defense responses has been hampered by numerous reports of comparable systems with contradictory interpretations of CRP as a stimulator, suppressor, or benign contributor to such processes. These discrepancies may be explained in part by the existence of a naturally occurring CRP isoform, termed modified CRP (i.e., mCRP), that is expressed when CRP subunits are dissociated into monomeric structures. The free mCRP subunit undergoes a non-proteolytic conformational change that has unique solubility, antigenicity, and bioactivity compared to the subunits that remain associated in the native, pentameric CRP molecule (i.e., pCRP). As specific reagents have been developed to identify and quantify mCRP, it has become apparent that this isoform can be formed spontaneously in calcium-free solutions. Furthermore, mCRP can be expressed on perturbed cell membranes with as little as 24–48 h incubation in tissue culture. Because mCRP has the same size as pCRP subunits as evaluated by SDS-PAGE, its presence in a pCRP reagent would not be apparent using this technique to evaluate purity. Finally, because many antibody reagents purported to be specific for “CRP” contains some, or substantial specificity to mCRP, antigen-detection techniques using such reagents may fail to distinguish the specific CRP isoform detected. All these caveats concerning CRP structures and measurements suggest that the aforementioned contradictory studies may reflect to some extent on distinctive bioactivities of mCRP rather than on pCRP. To provide a reliable, abundant supply of mCRP for separate and comparable studies, a recombinant protein was engineered and expressed in E. coli (i.e., recombinant mCRP or rmCRP). Synthesized protein was produced as inclusion bodies which proved difficult to solubilize for purification and characterization. Herein, we describe a method using anhydride reagents to effectively solubilize rmCRP and allow for chromatographic purification in high yield and free of contaminating endotoxin. Furthermore, the purified rmCRP reagent represents an excellent comparable protein to the biologically produced mCRP and as a distinctive reagent from pCRP. Deciphering the true function of CRP in both health and disease requires a knowledge, understanding, and reliable supply of each of its structures so to define the distinctive effects of each on the body’s response to tissue damaging events.

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Section: Discussionmentioning

confidence: 59%

Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification

Potempa

Yao

et al. 2015

Biophys. Rep.

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“…The secondary structure of a protein is the geometric structure caused by intermolecular and intramolecular hydrogen bonding of the amide groups [ 38 ]. Fig.…”

Section: Resultsmentioning

confidence: 99%

Investigating the Conformation of S100β Protein Under Physiological Parameters Using Computational Modeling: A Clue for Rational Drug Design

Tiburu

Issah

Darko

et al. 2018

TOBEJ

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Background:Physiochemical factors such as temperature, pH and cofactors are well known parameters that confer conformational changes in a protein structure. With S100β protein being a metal binding brain-specific receptor for both extracellular and intracellular functions, a change in conformation due to the above-mentioned factors, can compromise their cellular functions and therefore result in several pathological conditions such as Alzheimer’s disease, Ischemic stroke, as well as Myocardial Infarction.Objective:The studies conducted sought to elucidate the effect of these physiological factors on the conformational dynamics of S100β protein using computational modeling approaches.Method:Temperature-dependent and protein-cofactor complexes molecular dynamics simulations were conducted by varying the temperature from 100 to 400K using GROMACS 5.0.3. Additionally, the conformational dynamics of the protein was studied by varying the pH at 5.0, 7.4 and 9.0 using Ambertools17. This was done by preparing the protein molecule, solvating and minimizing its energy level as well as heating it to the required temperature, equilibrating and simulating under desired conditions (NVT and NPT ensembles).Results:The results show that the protein misfolds as a function of increasing temperature with alpha helical content at 100K and 400K being 57.8% and 43.3%, respectively. However, the binding sites of the protein was not appreciably affected by temperature variations. The protein displayed high conformational instability in acidic medium (pH ~5.0). The binding sites of Ca2+, Mg2+ and Zn2+ were identified and each exhibited different groupings of the secondary structural elements (binding motifs). The secondary structure analysis revealed different conformational changes with the characteristic appearance of two beta hairpins in the presence of Zn2+and Mg2+.Conclusion:High temperatures, different cofactors and acidic pH confer conformational changes to the S100β structure and these results may inform the design of novel drugs against the protein.

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“…Extracellular proteins mainly contain disulfides [116] and are exposed to a higher level of ROS in general [117]. Modification of the disulfides in receptors and plasma proteins is involved in protein stability [118], protein oligomerization [119], the transformation of biological function [120,121] and receptor-ligand interaction [122]. A similar interplay between ROS signaling, ligand recognition and protein-protein interaction is imaginable for AOC3 and ZAG, which could restrict the inhibitory function of ZAG.…”

Section: Discussionmentioning

confidence: 99%

Zinc-α2-Glycoprotein Is An Inhibitor Of Amine Oxidase Copper-Containing 3

Romauch¹

2019

Preprint

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6Zinc-alpha2-glycoprotein (ZAG) is a major plasma protein whose levels increase in 7 chronic energy-demanding diseases and thus serves as an important clinical biomarker 8 in the diagnosis and prognosis of the development of cachexia. Current knowledge 9 suggests that ZAG mediates progressive weight loss through β-adrenergic signaling in 10 adipocytes, resulting in the activation of lipolysis and fat mobilization. Here, through 11 crosslinking experiments, amine oxidase copper-containing 3 (AOC3) is identified as a 12 novel ZAG binding partner. AOC3 -also known as vascular adhesion protein 1 (VAP-1) 13 and semicarbazide sensitive amine oxidase (SSAO) -deaminates primary amines, 14 thereby generating the corresponding aldehyde, H2O2 and HN3. It is an ectoenzyme 15 largely expressed by adipocytes and induced in endothelial cells during inflammation. H2O2 has an insulinogenic effect. The observations described here suggest that ZAG acts 18 as an allosteric inhibitor of AOC3 and interferes with the associated pro-inflammatory 19 and anti-lipolytic functions. Thus, inhibition of the deamination of lipolytic hormone 20 octopamine by AOC3 represents a novel mechanism by which ZAG might stimulate 21 lipolysis. Furthermore, experiments involving overexpression of recombinant ZAG 22 reveal that its glycosylation is co-regulated by oxygen availability and that the pattern of 23 glycosylation affects its inhibitory potential. The newly identified protein interaction 24 2 between AOC3 and ZAG highlights a previously unknown functional relationship, which 25 may be relevant to inflammation, energy metabolism and the development of cachexia. 26 27 93 provide much-needed insight into the mechanism of ZAG function and stimulate future work 94 in basic and clinical research on ZAG.95 96 5 2 Results 97 2.1 ZAG binds to ectoenzyme AOC3 98To attempt to identify ZAG interaction partners, purified recombinant ZAG and freshly 99 prepared adipocyte plasma membranes were co-incubated and any physical interactions 100 between them were stabilized by a photoactivatable crosslinker molecule (Fig. 11). Both 101 human and murine ZAG (without leader sequence) were produced in E. coli after cloning in 102 the expression plasmid pGEX-6P-2 and affinity purified by GST (glutathione-S-transferase)-103 tag. Both purified human and mouse proteins (GST-hZAG and GST-mZAG, respectively) and 104 GST-tag aloneserving as a controlwere labeled with the photoactivatable crosslinker 105

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Computational Analysis of C-Reactive Protein for Assessment of Molecular Dynamics and Interaction Properties

Cited by 10 publications

References 83 publications

Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification

Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification

Investigating the Conformation of S100β Protein Under Physiological Parameters Using Computational Modeling: A Clue for Rational Drug Design

Zinc-α2-Glycoprotein Is An Inhibitor Of Amine Oxidase Copper-Containing 3

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