BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy

Micsonai, András; Bulyáki, Éva; Kardos, József

doi:10.1007/978-1-0716-0892-0_11

Cited by 76 publications

(58 citation statements)

References 16 publications

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“…CD analysis of our construct confirms the conservation of the typical chemokine fold ( Figure 4E , NRMSD 0.01858). The secondary structure distributions predicted from the fit using Bestsel ( Micsonai et al, 2015 ; Micsonai et al, 2018 ; Micsonai et al, 2021 ) are consistent with the solution state NMR structure 1EOT ( Crump et al, 1998 ) ( Figure 4F ).…”

Section: Resultssupporting

confidence: 72%

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Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling

Aalst

Wylie

2021

Front. Mol. Biosci.

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Cholesterol as an allosteric modulator of G protein-coupled receptor (GPCR) function is well documented. This quintessential mammalian lipid facilitates receptor–ligand interactions and multimerization states. Functionally, this introduces a complicated mechanism for the homeostatic modulation of GPCR signaling. Chemokine receptors are Class A GPCRs responsible for immune cell trafficking through the binding of endogenous peptide ligands. CCR3 is a CC motif chemokine receptor expressed by eosinophils and basophils. It traffics these cells by transducing the signal stimulated by the CC motif chemokine primary messengers 11, 24, and 26. These behaviors are close to the human immunoresponse. Thus, CCR3 is implicated in cancer metastasis and inflammatory conditions. However, there is a paucity of experimental evidence linking the functional states of CCR3 to the molecular mechanisms of cholesterol–receptor cooperativity. In this vein, we present a means to combine codon harmonization and a maltose-binding protein fusion tag to produce CCR3 from E. coli. This technique yields ∼2.6 mg of functional GPCR per liter of minimal media. We leveraged this protein production capability to investigate the effects of cholesterol on CCR3 function in vitro. We found that affinity for the endogenous ligand CCL11 increases in a dose-dependent manner with cholesterol concentration in both styrene:maleic acid lipid particles (SMALPs) and proteoliposomes. This heightened receptor activation directly translates to increased signal transduction as measured by the GTPase activity of the bound G-protein α inhibitory subunit 3 (Gαi3). This work represents a critical step forward in understanding the role of cholesterol-GPCR allostery in regulation of signal transduction.

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Section: Resultssupporting

confidence: 72%

“…(E) CD spectra of CCL11 (blue) compared to the fit (orange) and the residual fit (purple). The spectrum was fit using Bestsel ( Micsonai et al, 2015 ; Micsonai et al, 2018 ; Micsonai et al, 2021 ). (F) Bestsel-predicted percent secondary structure for the CCL11 construct compared to the NMR structure (PDB ID 1EOT) ( Crump et al, 1998 ).…”

Section: Resultsmentioning

confidence: 99%

Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling

Aalst

Wylie

2021

Front. Mol. Biosci.

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“…Oxidized DTT (trans-4,5-dihydroxy-1,2-dithiane) served as a control compound, being identical to DTT, except for two thiol groups forming an intramolecular S-S bond. The compounds were added to the final concentration of 2.5 mM, the highest concentration possible without causing a significant increase in the UV light absorption (Micsonai et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

Spike protein disulfide disruption as a potential treatment for SARS-CoV-2

Grishin

Dolgova

Harms

et al. 2021

Preprint

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The coronaviral pandemic is exerting a tremendously detrimental impact on global health, quality of life and the world economy, emphasizing the need for effective medications for current and future coronaviral outbreaks as a complementary approach to vaccines. The Spike protein, responsible for cell receptor binding and viral internalization, possesses multiple disulfide bonds raising the possibility that disulfide-reducing agents might disrupt Spike function, prevent viral entry and serve as effective drugs against SARS-CoV-2. Here we show the first experimental evidence that reagents capable of reducing disulfide bonds can inhibit viral infection in cell-based assays. Molecular dynamics simulations of the Spike receptor-binding domain (RBD) predict increased domain flexibility when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the surface loop, comprised of residues 456-490, which interacts with the Spike cell receptor ACE2. Consistent with this finding, the addition of exogenous disulfide bond reducing agents affects the RBD secondary structure, lowers its melting temperature from 52 to 36-39°C and decreases its binding affinity to ACE2 by two orders of magnitude at 37°C. Finally, the reducing agents dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) inhibit viral replication at high µM – low mM levels with a negligible effect on cell viability at these concentrations. The antiviral effect of monothiol-based reductants N-Acetyl-L-cysteine (NAC) and reduced glutathione (GSH) was not observed due to decreases in cell viability. Our research demonstrates the clear potential for medications that disrupt Spike disulfides as broad-spectrum anticoronaviral agents and as a first-line defense against current and future outbreaks.

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“…SRCD spectra were recorded at the DISCO SRCD beamline of a SOLEIL Synchrotron (Gif-sur-Yvette, France). Experimental parameters were set by following the guidelines of Micsonai et al [36]. Protein concentration was~5 mg/mL in 10 mM Na-phosphate, pH 7.4, using a 12 µm CaF 2 cell (Hellma GmbH, Müllcheim, Germany).…”

Section: Spectroscopy Measurementsmentioning

confidence: 99%

Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14

Murvai

Kalmár

Szabó

et al. 2021

IJMS

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Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana’s Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.

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BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy

Cited by 76 publications

References 16 publications

Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling

Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling

Spike protein disulfide disruption as a potential treatment for SARS-CoV-2

Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14

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