A constant-volume ultrafiltration technique for the calculation of equilibrium binding data

1978 ◽  
Vol 543 (3) ◽  
pp. 397-402 ◽  
Author(s):  
Barry W.A. Williamson ◽  
Richard C. Strange ◽  
Iain W. Percy-Robb
Keyword(s):  
Constant Volume ◽  
Equilibrium Binding ◽  
Binding Data

scholarly journals Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

2021 ◽  
Author(s):  
Jaryd R Sullivan ◽  
Andreanne Lupien ◽  
Elias Kalthoff ◽  
Claire Hamela ◽  
Lorne Taylor ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Mycobacterium Abscessus ◽  
Trna Synthetases ◽  
Equilibrium Binding ◽  
Binding Data ◽  
Clp Proteases ◽  
Vitro Data ◽  
In Vitro Data

Certain aminoacyl-tRNA synthetases developed a proofreading mechanism to ensure aminoacylation of tRNAs with cognate amino acids. Epetraborole (EPT) was identified as an inhibitor of the leucyl-tRNA synthetase (LeuRS) editing site in Mycobacterium abscessus. EPT displayed enhanced activity against M. abscessus over Mycobacterium tuberculosis. Crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar Kd. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed the non-proteinogenic amino acid norvaline, leucine residues in proteins were replaced by norvaline, inducing expression of GroEL chaperonins and Clp proteases. In vitro data revealed that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. The combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection.

scholarly journals Resolution of the Equilibrium Constant for the T State → RState Conformational Change of Human Hemoglobin into Endothermic and Exothermic Component Reactions

2019 ◽  
Author(s):  
Francis Knowles ◽  
Douglas Magde
Keyword(s):  
Equilibrium Constant ◽  
Whole Blood ◽  
Binding Constant ◽  
Binary Complex ◽  
Human Hemoglobin ◽  
Conformation Change ◽  
Unknown Quantity ◽  
Equilibrium Binding ◽  
Binding Data ◽  
T State

<p> The dimensionless equilibrium constant for the allosteric conformation change, K<sub>ΔC</sub> = 0.02602 (Knowles & Magde, linked ms 2) following binding of O<sub>2</sub> by α-chains in <sup>T</sup>state Hb<sub>4</sub>/BPG (whole blood under standard conditions) is shown to be comprised of: (i) an endothermic change in conformation, from <sup>T</sup>state to <sup>R</sup>state, of 24.3 kJ/mol; (ii) exothermic conversion of <sup>T</sup>state <sup>T</sup>αO<sub>2</sub>-chains to <sup>R</sup>state <sup>R</sup>αO<sub>2</sub>-chains of -13.8 kJ/mol; (iii)exothermic binding of BPG by R-states. Eq. (1) defines the component steps whereby the <sup>T</sup>state conformation is converted to the <sup>R</sup>state conformation.</p> <p>ΔG<sup>o</sup>(<sup>R</sup>(Hb<sub>4</sub>), BPG) describes the endothermic decomposition of the binary complex, <sup>T</sup>Hb<sub>4</sub>/BPG into <sup>R</sup>Hb<sub>4</sub> and BPG, equal to + 33.7 kJ/mol (DeBruin et al. (1973). J. Biol. Chem. <u>248</u>, 2774-2777). ΔG<sup>o</sup> for the equilibrium constant for ΔG<sup>O</sup>(K<sub>ΔC</sub>) and Σ ΔG<sup>o</sup> for binding of O<sub>2</sub> by the pair of equivalent <sup>T</sup>state α-chains, ΔG<sup>O</sup>(<sup>T</sup>α<sup>*</sup>O<sub>2</sub>), + 9.41 kJ/mol and – 49.6 kJ/mol, respectively, are determined by fitting of O<sub>2</sub> equilibrium binding data to the Perutz-Adair equation. ΔG<sup>o</sup> for reaction of a pair of equivalent <sup>R</sup>state α-chains with O<sub>2</sub>, ΔG<sup>O</sup>(<sup>R</sup>αO<sub>2</sub>), was estimated from the known affinity of myoglobin for O<sub>2</sub> at 37<sup>o</sup>C (Theorell H. (1936). Biochem. Z., <u>268</u>, 73-81), -63.4 kJ/mol. The unknown quantity, ∆G<sup>O</sup>(<sup>R</sup>(HbO<sub>2</sub>)<sub>4</sub>/BPG), was obtained by solving Eq. (1), being -10.5 kJ/mol, K (<sup>R</sup>(HbO<sub>2</sub>)<sub>4</sub>/BPG) = 58.4 L/mol. The value of the equilibrium constant for binding BPG to R-state conformations represents 0.0073% of the value of the binding constant of BPG to <sup>T</sup>state conformations: 800,000 L/mol. The value of K<sub>ΔC</sub>; (i) accounts for the ability of O<sub>2</sub> to escape, virtually unhindered from rbcs and (ii) provides a biophysical basis for manifestation of high resting rates of metabolism in warm blooded species.</p>

scholarly journals Salt bridges gate α-catenin activation at intercellular junctions

2018 ◽  
Vol 29 (2) ◽  
pp. 111-122 ◽  
Author(s):  
Samantha Barrick ◽  
Jing Li ◽  
Xinyu Kong ◽  
Alokananda Ray ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Mechanical Activation ◽  
Conformational Changes ◽  
Intercellular Junctions ◽  
Salt Bridge ◽  
Salt Bridges ◽  
Equilibrium Binding ◽  
Binding Data ◽  
Dynamics Simulations ◽  
Intercellular Adhesions

Molecular dynamics simulations, equilibrium binding measurements, and fluorescence imaging reveal the influence of a key salt bridge in the mechanical activation of α-catenin at intercellular adhesions. Simulations reveal possible α-catenin conformational changes underlying experimental fluorescence and equilibrium binding data.

scholarly journals Resolution of the Equilibrium Constant for the T State → RState Conformational Change of Human Hemoglobin into Endothermic and Exothermic Component Reactions

2019 ◽  
Author(s):  
Francis Knowles ◽  
Douglas Magde
Keyword(s):  
Equilibrium Constant ◽  
Whole Blood ◽  
Binding Constant ◽  
Binary Complex ◽  
Human Hemoglobin ◽  
Conformation Change ◽  
Unknown Quantity ◽  
Equilibrium Binding ◽  
Binding Data ◽  
T State

<p> The dimensionless equilibrium constant for the allosteric conformation change, K<sub>ΔC</sub> = 0.02602 (Knowles & Magde, linked ms 2) following binding of O<sub>2</sub> by α-chains in <sup>T</sup>state Hb<sub>4</sub>/BPG (whole blood under standard conditions) is shown to be comprised of: (i) an endothermic change in conformation, from <sup>T</sup>state to <sup>R</sup>state, of 24.3 kJ/mol; (ii) exothermic conversion of <sup>T</sup>state <sup>T</sup>αO<sub>2</sub>-chains to <sup>R</sup>state <sup>R</sup>αO<sub>2</sub>-chains of -13.8 kJ/mol; (iii)exothermic binding of BPG by R-states. Eq. (1) defines the component steps whereby the <sup>T</sup>state conformation is converted to the <sup>R</sup>state conformation.</p> <p>ΔG<sup>o</sup>(<sup>R</sup>(Hb<sub>4</sub>), BPG) describes the endothermic decomposition of the binary complex, <sup>T</sup>Hb<sub>4</sub>/BPG into <sup>R</sup>Hb<sub>4</sub> and BPG, equal to + 33.7 kJ/mol (DeBruin et al. (1973). J. Biol. Chem. <u>248</u>, 2774-2777). ΔG<sup>o</sup> for the equilibrium constant for ΔG<sup>O</sup>(K<sub>ΔC</sub>) and Σ ΔG<sup>o</sup> for binding of O<sub>2</sub> by the pair of equivalent <sup>T</sup>state α-chains, ΔG<sup>O</sup>(<sup>T</sup>α<sup>*</sup>O<sub>2</sub>), + 9.41 kJ/mol and – 49.6 kJ/mol, respectively, are determined by fitting of O<sub>2</sub> equilibrium binding data to the Perutz-Adair equation. ΔG<sup>o</sup> for reaction of a pair of equivalent <sup>R</sup>state α-chains with O<sub>2</sub>, ΔG<sup>O</sup>(<sup>R</sup>αO<sub>2</sub>), was estimated from the known affinity of myoglobin for O<sub>2</sub> at 37<sup>o</sup>C (Theorell H. (1936). Biochem. Z., <u>268</u>, 73-81), -63.4 kJ/mol. The unknown quantity, ∆G<sup>O</sup>(<sup>R</sup>(HbO<sub>2</sub>)<sub>4</sub>/BPG), was obtained by solving Eq. (1), being -10.5 kJ/mol, K (<sup>R</sup>(HbO<sub>2</sub>)<sub>4</sub>/BPG) = 58.4 L/mol. The value of the equilibrium constant for binding BPG to R-state conformations represents 0.0073% of the value of the binding constant of BPG to <sup>T</sup>state conformations: 800,000 L/mol. The value of K<sub>ΔC</sub>; (i) accounts for the ability of O<sub>2</sub> to escape, virtually unhindered from rbcs and (ii) provides a biophysical basis for manifestation of high resting rates of metabolism in warm blooded species.</p>

scholarly journals Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1411
Author(s):  
Giacomo Parisi ◽  
Ida Freda ◽  
Cécile Exertier ◽  
Cristina Cecchetti ◽  
Elena Gugole ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Structural Response ◽  
Structural Elements ◽  
X Ray ◽  
In Silico Docking ◽  
Equilibrium Binding ◽  
Binding Data ◽  
Minor Population ◽  
Solvent Molecules ◽  
A Minor

The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein–substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP–DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound.

scholarly journals How to measure and evaluate binding affinities

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Inga Jarmoskaite ◽  
Ishraq AlSadhan ◽  
Pavanapuresan P Vaidyanathan ◽  
Daniel Herschlag
Keyword(s):  
Rna Binding ◽  
Rna Binding Protein ◽  
Mechanistic Models ◽  
Binding Affinities ◽  
Measurement Reliability ◽  
High Quality ◽  
Quantitative Measurements ◽  
Equilibrium Binding ◽  
Binding Data ◽  
Current Standards

Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it especially timely to evaluate the current standards for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concentration regime were not documented, making it impossible to determine measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biological interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equilibrium binding measurements. We apply this framework and explain underlying fundamental concepts through experimental examples with the RNA-binding protein Puf4.

scholarly journals High-affinity binding of lower-density lipoproteins to chicken oocyte membranes

1981 ◽  
Vol 196 (2) ◽  
pp. 481-488 ◽  
Author(s):  
S A Krumins ◽  
T F Roth
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Cell Types ◽  
Low Density ◽  
Binding Assays ◽  
Equilibrium Binding ◽  
Binding Data ◽  
Lipoprotein Binding ◽  
Oocyte Membrane ◽  
Competitive Binding Assays

Oocyte membrane fragments bind specifically radioiodinated VLD lipoprotein (very-low density lipoprotein) and LD lipoprotein (low-density lipoprotein). Competitive binding assays showed 2-3 times more VLD lipoprotein than LD lipoprotein bound at 4 degrees C. Equilibrium-binding data revealed the presence of one class of non-interacting sites for VLD lipoprotein (kD 12 microgram/ml) and co-operative binding for LD lipoprotein. The binding of VLD lipoprotein showed a distinct pH maximum at 5.3, whereas an indistinct maximum at about pH 7.3 was observed for LD lipoprotein. Unlabelled VLD lipoprotein did compete with 125I-labelled LD lipoprotein binding, but unlabelled LD lipoprotein did not compete with 125I-labelled VLD lipoprotein binding. VLD lipoprotein binding was inhibited by HD lipoprotein (high-density lipoprotein), but not by lysozyme, collagen, poly-L-lysine or poly-L-arginine; LD lipoprotein binding was inhibited by lysozyme and collagen, but not by HD lipoprotein. On the basis of these studies, we suggest that: (1) VLD lipoprotein and LD lipoprotein enter the oocytes by a receptor-mediated transport mechanism; (2) the receptors for VLD lipoprotein and LD lipoprotein are distinct; and (3) the binding of LD lipoprotein to chicken oocyte membranes differs from that to other cell types.

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