Imaging and kinetics of the bimolecular complex formed by the tumor suppressor p53 with ubiquitin ligase COP1 as studied by atomic force microscopy and surface plasmon resonance

Moscetti, Ilaria; Bizzarri, Anna Rita; Cannistraro, Salvatore

doi:10.2147/ijn.s152214

Cited by 17 publications

(10 citation statements)

References 44 publications

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“…The model used for ligand leakage (Ltrue(ttrue)) during dissociation consisted of a constant part (basal value: Ltrue(0true)) and an exponentially decaying function which is dependent on time (t); Ltrue(ttrue)=Ltrue(0true)+Ltrue(0true)e−kleakt; where, kleak, is the leakage rate. The simulation profiles were consistent with that of the earlier reported experimental profiles [7,8,32].…”

Section: Resultssupporting

confidence: 90%

Isothermal titration calorimetry and surface plasmon resonance analysis using the dynamic approach

Krishnamoorthy¹,

Alluvada

Sherieff³

et al. 2020

Biochemistry and Biophysics Reports

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Biophysical techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants. Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2′-CMP + RNASE and BH3I-1 + hBCLXL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately.

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Section: Resultssupporting

confidence: 90%

Isothermal titration calorimetry and surface plasmon resonance analysis using the dynamic approach

Krishnamoorthy¹,

Alluvada

Sherieff³

et al. 2020

Biochemistry and Biophysics Reports

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show abstract

“…, is the leakage rate. The simulation profiles were consistent with that of the earlier reported experimental profiles [6,7,31].…”

Section: Resultssupporting

confidence: 90%

ITC and SPR Analysis Using Dynamic Approach

Krishnamoorthy¹,

Krishanmoorthy

Alluvada

et al. 2019

Preprint

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Biophysical techniques such as Isothermal Calorimetry (ITC) and Surface Plasmon Resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants.Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2'-CMP + RNASE and BH3I-1 + hBCL XL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately.

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“…The StrepMAB-Immo™ (Iba, Goettingen, Germany) was immobilized onto the CM5 sensor chip surface (GE Healthcare) by using the Amine Coupling kit (GE Healthcare) and the HBS-EP + running buffer (GE Healthcare), following the procedure reported in refs. 50,51 . Briefly, after activation of CM5 sensor chip, a solution of StrepMAB-Immo™ (50 μg/mL) in immobilization buffer (10 mM sodium acetate pH 5.0, GE Healthcare) was fluxed over the reactive matrix at a flow rate of 10 μL/min for 7 minutes.…”

Section: Methodsmentioning

confidence: 99%

Interaction of human hemoglobin and semi-hemoglobins with the Staphylococcus aureus hemophore IsdB: a kinetic and mechanistic insight

Gianquinto

Moscetti

Bei

et al. 2019

Sci Rep

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Among multidrug-resistant bacteria, methicillin-resistant Staphylococcus aureus is emerging as one of the most threatening pathogens. S. aureus exploits different mechanisms for its iron supply, but the preferred one is acquisition of organic iron through the expression of hemoglobin (Hb) receptors. One of these, IsdB, belonging to the Isd (Iron-Regulated Surface Determinant) system, was shown to be essential for bacterial growth and virulence. Therefore, interaction of IsdB with Hb represents a promising target for the rational design of a new class of antibacterial molecules. However, despite recent investigations, many structural and mechanistic details of complex formation and heme extraction process are still elusive. By combining site-directed mutagenesis, absorption spectroscopy, surface plasmon resonance and molecular dynamics simulations, we tackled most of the so far unanswered questions: (i) the exact complex stoichiometry, (ii) the microscopic kinetic rates of complex formation, (iii) the IsdB selectivity for binding to, and extracting heme from, α and β subunits of Hb, iv) the role of specific amino acid residues and structural regions in driving complex formation and heme transfer, and (v) the structural/dynamic effect played by the hemophore on Hb. The ability of bacterial pathogens to establish infections relies on the adaptation of bacterial metabolism to the environment found within the host, which is often severely nutrient-restricted and a site for competition with commensal microorganisms and other pathogens 1,2. A well-characterized mechanism of adaptation to the nutritional environment of the host is represented by the expression of redundant iron-acquisition systems, both in Gram + and Grambacteria, to provide iron supply to support invasion and proliferation 3. Iron is an essential nutrient for both the pathogen and the host, but also a toxic element due to its involvement in Fenton chemistry and Haber-Weiss reactions that generate reactive oxygen species (ROS) 4. For this reason, vertebrates have put in place strategies to maintain very low concentrations of free iron in body fluids (down to 10 −18-10 −24 M) 4,5 achieving two goals, the first being limiting toxicity and the second creating an iron-restricted environment for pathogens 6,7. In S. aureus, iron scavenging is achieved by at least three different mechanisms: i) acquisition of inorganic iron through production of siderophores, ii) expression of hemoglobin (Hb) receptors, and iii) acquisition of inorganic iron through transporters 4. Heme is the preferred iron source during the initial phase of infection 8

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Imaging and kinetics of the bimolecular complex formed by the tumor suppressor p53 with ubiquitin ligase COP1 as studied by atomic force microscopy and surface plasmon resonance

Cited by 17 publications

References 44 publications

Isothermal titration calorimetry and surface plasmon resonance analysis using the dynamic approach

Isothermal titration calorimetry and surface plasmon resonance analysis using the dynamic approach

ITC and SPR Analysis Using Dynamic Approach

Interaction of human hemoglobin and semi-hemoglobins with the Staphylococcus aureus hemophore IsdB: a kinetic and mechanistic insight

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