Impact of protein and ligand impurities on ITC-derived protein–ligand thermodynamics

Grüner, Stefan; Neeb, M.; Barandun, Luzi Jakob; Sielaff, Frank; Hohn, Christoph; Kojima, Satoko; Steinmetzer, Torsten; Diederich, François; Klebe, G.

doi:10.1016/j.bbagen.2014.04.018

Cited by 23 publications

(22 citation statements)

References 37 publications

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“…35 The ITC results shown in Figure 4 were corrected for dilution effects by subtracting the reference data measured in identical series of injections into buffer, and fit the Table 2. More specifically, electrostatic binding between SOD and Pb(Ac) 2 was confirmed by negative ΔH and positive ΔS values.…”

Section: Methodsmentioning

confidence: 99%

Molecular Mechanism of Lead-Induced Superoxide Dismutase Inactivation in Zebrafish Livers

Zhang¹,

Yang²,

Liu³

et al. 2014

J. Phys. Chem. B

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Lead toxicity has been proved to be related with inducing oxidative stress of organisms and causing inactivation of antioxidant enzymes, the mechanism of which remains unknown. This study investigated and compared superoxide dismutase (Cu/Zn SOD) activity inhibited in lead-treated zebrafish livers and explored the mechanism of SOD inactivation by lead at the molecular level using multiple spectroscopic techniques, isothermal titration calorimetric (ITC) measurement, molecular docking study and ICP-AES detection. Results showed lead exposure decreased SOD activities in zebrafish livers due to direct interactions between lead and SOD, resulting in conformational and functional changes of the enzyme. To be specific, Studies at the molecular level indicated that lead bound into the active site channel of SOD, hindered the path of the catalytic substrate (O(2)(-•)), damaged its skeleton conformation and secondary structure, and interacted with the enzymatically related residue (Arg 141) through electrostatic forces (ΔH < 0, ΔS > 0), and caused the release of Cu(2+) and Zn(2+) from the catalytic pocket of SOD. This work shows a correlation between results on organismal and molecular levels, and obtains a possible model hypothesizing mechanisms of lead toxicity using in vitro experiments instead of in vivo ones.

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Section: Methodsmentioning

confidence: 99%

Molecular Mechanism of Lead-Induced Superoxide Dismutase Inactivation in Zebrafish Livers

Zhang¹,

Yang²,

Liu³

et al. 2014

J. Phys. Chem. B

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“…Fluorescence spectroscopy and thermophoresis require labeling Lw ith af luorophore,and affects Lsbinding to Pand, thus,the accuracy of the K d measurements. [6] An inherent source of inaccuracyinK d values determined by direct mass-spectrometry (MS) is ashift of the equilibrium in Equation 1during the transfer of L, P, and PL from the liquid or solid phase to the gas phase,a nd during their ionization. Biosensoric techniques require the immobilization of either L or Ponto asensor surface,that also affects binding and, thus, the accuracy of the K d measurements.…”

mentioning

confidence: 99%

“…[5] Isothermal titration calorimetry does not require labeling or immobilization but has another source of inaccuracy: the heat of side reactions (e.g.,b inding of Lt oh igh-concentration impurities in P, binding of PtoP ,and solvation of protons released upon Ls binding to P). [6] An inherent source of inaccuracyinK d values determined by direct mass-spectrometry (MS) is ashift of the equilibrium in Equation 1during the transfer of L, P, and PL from the liquid or solid phase to the gas phase,a nd during their ionization. [7] TheaccuracyofK d determination with sizeexclusion chromatography is affected by inevitable adsorption of L, P, and PL onto the stationary phase.…”

mentioning

confidence: 99%

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

Sisavath

Rukundo

Leblanc³

et al. 2019

Angew Chem Int Ed

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Current practical methods for finding the equilibrium dissociation constant, K d ,o fp rotein-small molecule complexes have inherent sources of inaccuracy.I ntroduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy.C onceptually,as hort plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in ac apillary,c ausing fast transient incomplete separation of the complex from the unbound small molecule.Asuperposition of signals from these two components is measured near the capillary exit and used to calculate af raction of unbound small molecule,which,inturn, is used to calculate K d . Herein the validity of ACTIS is proven theoretically,i ts accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become ar eference-standardm ethod for determining K d values of protein-small molecule complexes.Reversible binding of proteins (P) to small-molecule ligands (L) plays an important role in the regulation of cellular processes. [1] In addition, most therapeutic targets are proteins, [2] and drugs are developed to form stable PL complexes with them:Complex stability is characterized by the equilibrium dissociation constant K d ,which is defined as:

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“…It is possible that loss of function due to the refolding procedure, incomplete ligand removal or sample aggregation during titration experiments contributed to a decrease in the final calculated N values. However, all binding curves with native or refolded protein could be fit using one‐site binding equations and low N values should not adversely affect the affinity constants reported here …”

Section: Resultsmentioning

confidence: 99%

Transport capabilities of environmental Pseudomonads for sulfur compounds

et al. 2017

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Sulfur is an essential element in plant rhizospheres and microbial activity plays a key role in increasing the biological availability of sulfur in soil environments. To better understand the mechanisms facilitating the exchange of sulfur-containing molecules in soil, we profiled the binding specificities of eight previously uncharacterized ABC transporter solute-binding proteins from plant-associated Pseudomonads. A high-throughput screening procedure indicated eighteen significant organosulfur binding ligands, with at least one high-quality screening hit for each protein target. Calorimetric and spectroscopic methods were used to validate the best ligand assignments and catalog the thermodynamic properties of the protein-ligand interactions. Two novel highaffinity ligand-binding activities were identified and quantified in this set of solute-binding proteins. Bacteria were cultured in minimal media with screening library components supplied as the sole sulfur sources, demonstrating that these organosulfur compounds can be metabolized and confirming the relevance of ligand assignments. These results expand the set of experimentally validated ligands amenable to transport by this ABC transporter family and demonstrate the complex range of protein-ligand interactions that can be accomplished by solute-binding proteins. Characterizing new nutrient import pathways provides insight into Pseudomonad metabolic capabilities which can be used to further interrogate bacterial survival and participation in soil and rhizosphere communities. Statement: We report protein-ligand interactions for eight solute-binding proteins of organosulfur compounds, including two previously unknown binding activities. These patterns of activity expand the range of small molecules recognized by the methioninebinding protein family of ABC transporters and provide additional insight into the transport and metabolic capabilities of Pseudomonads.Grant sponsors:

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Impact of protein and ligand impurities on ITC-derived protein–ligand thermodynamics

Cited by 23 publications

References 37 publications

Molecular Mechanism of Lead-Induced Superoxide Dismutase Inactivation in Zebrafish Livers

Molecular Mechanism of Lead-Induced Superoxide Dismutase Inactivation in Zebrafish Livers

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

Transport capabilities of environmental Pseudomonads for sulfur compounds

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