scholarly journals Evidence for a kinetic heterogeneity in ligand binding to R-state haemoglobin Kempsey [Asp-G1(99) β→Asn]

1986 ◽  
Vol 238 (2) ◽  
pp. 353-357
Author(s):  
M Coletta ◽  
T Brittain ◽  
M Brunori
Keyword(s):  
Ligand Binding ◽  
Time Course ◽  
Flash Photolysis ◽  
Activation Parameters ◽  
Binding Kinetics ◽  
Slow Phase ◽  
Kinetic Properties ◽  
Dissociation Kinetics ◽  
Binding Isotherms ◽  
O2 Binding

Thermodynamic and kinetic properties of O2 and CO binding to haemoglobin (Hb) Kempsey [Asp-G1(99) beta----Asn] were investigated and the activation parameters for the two ligands were determined. At every temperature the O2-binding isotherms display a weak co-operativity, n ranging between 1.1 and 1.2, and dissociation kinetics show a single-exponential behaviour. O2-binding kinetics were studied at 25 degrees C by temperature jump and are characterized at each saturation (from Y = 0.31 to Y = 1.0) by two processes, a fast bimolecular one and a slow monomolecular one (tau -1 = 20 s-1), which contributes to approx. 30% of the whole relaxation amplitude at every Y. CO-binding kinetics to Hb Kempsey were followed at several temperatures by flash photolysis and stopped flow. The process is biphasic, as reported elsewhere [Bunn, Wohl, Bradley, Cooley & Gibson (1974) J. Biol. Chem. 249, 7402-7409], and the relative contributions of the two bimolecular rates to the whole process are only slightly affected by temperature. On taking account for the fraction of dimers at every protein concentration, the slow phase corresponds to approx. 50% of the ligand binding to tetramers. Correlation of these results with previous spectroscopic data leads to the hypothesis that the biphasic time course of CO binding may be attributed to alpha/beta heterogeneity of the R-state of tetrameric Hb Kempsey.

scholarly journals The oligomeric state of thyroid receptor regulates hormone binding kinetics

2011 ◽  
Vol 210 (1) ◽  
pp. 125-134 ◽  
Author(s):  
Suzana T Cunha Lima ◽  
Edson D Rodrigues
Keyword(s):  
Ligand Binding ◽  
Half Life ◽  
Thyroid Hormone Receptor ◽  
Binding Pocket ◽  
Binding Kinetics ◽  
Hormone Binding ◽  
Oligomeric State ◽  
Dissociation Kinetics ◽  
Biphasic Kinetics ◽  
X Receptors

We previously reported that mutations in the thyroid hormone receptor (TR) surface that mediates dimer and heterodimer formation do not alter affinity for cognate hormone (triiodothyronine (T3)) yet dramatically enhance T3 association and dissociation rates. This study aimed to show that TR oligomeric state influences binding and dissociation kinetics. We performed binding assays using marked hormone (125I-T3) and TRs expressed and purified by different methods. We find that T3 associates with TRs with biphasic kinetics in solution; a rapid step (half-life ±0.1 h) followed by a slower second step (half-life ±5 h) and that purification of monomers suggests that biphasic kinetics are due to the presence of monomers and dimers in our preparations. In support of this idea, incubation of TR ligand binding domain monomers with corepressor peptide induces dimer formation and decreases association rates and T3 binds to, and dissociates from, a TRβ mutant that only forms dimers (TRβD355R) with slow single-phase kinetics. In addition, heterodimer formation with retinoid X receptors also influences ligand binding kinetics. Together, these results suggest that the dimer/heterodimer surface is allosterically coupled to the hormone binding pocket and that different interactions at this surface exert different effects on ligand binding that may be relevant for TR actions in the cell.

scholarly journals Ligand binding kinetics and dissociation of the human embryonic haemoglobins

1996 ◽  
Vol 315 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Oliver HOFMANN ◽  
Thomas BRITTAIN
Keyword(s):  
Ligand Binding ◽  
Flash Photolysis ◽  
Ph Dependence ◽  
Equilibrium Constants ◽  
General Scheme ◽  
Dissociation Rate ◽  
Oxygen Binding ◽  
Dissociation Kinetics ◽  
Oxygen Dissociation ◽  
Dissociation Rate Constants

The three human embryonic haemoglobins have been studied using a range of stopped-flow and flash photolysis experiments. The association and dissociation kinetics and equilibrium constants for the tetramer–dimer reactions of the deoxy and oxygenated forms have been investigated and found to be characterized by constants similar to those of the human adult protein. The rates of oxygen dissociation from the embryonic haemoglobins have been measured and appear to be responsible for the high oxygen-binding affinity associated with the embryonic proteins compared with the adult protein. The pH dependence of the oxygen dissociation rate constants also accounts for the rather unusual, previously described, Bohr effects characteristic of the embryonic haemoglobins. A general scheme has been developed coupling both the dimer–tetramer equilibria and ligand-binding steps observed following photolysis of the liganded forms of the human embryonic haemoglobins.

scholarly journals Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin

2021 ◽  
Vol 22 (5) ◽  
pp. 2740
Author(s):  
Stefania Abbruzzetti ◽  
Alex J. Barker ◽  
Irene Villar ◽  
Carmen Pérez-Rontomé ◽  
Stefano Bruno ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Rate Constants ◽  
Time Course ◽  
Flash Photolysis ◽  
Laser Flash Photolysis ◽  
Binding Constants ◽  
Laser Flash ◽  
High Reactivity ◽  
Nanosecond Laser ◽  
Ligand Rebinding

The recently identified nonsymbiotic hemoglobin gene MtGlb1-2 of the legume Medicago truncatula possesses unique properties as it generates four alternative splice forms encoding proteins with one or two heme domains. Here we investigate the ligand binding kinetics of MtGlb1-2.1 and MtGlb1-2.4, bearing two hemes and one heme, respectively. Unexpectedly, the overall time-course of ligand rebinding was unusually fast. Thus, we complemented nanosecond laser flash photolysis kinetics with data collected with a hybrid femtosecond–nanosecond pump–probe setup. Most photodissociated ligands are rebound geminately within a few nanoseconds, which leads to rates of the bimolecular rebinding to pentacoordinate species in the 108 M−1s−1 range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (kh ~ 105 s−1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (KH ~ 1). The rate constants for ligation and deligation of distal histidine to the heme are the highest reported for any plant or vertebrate globin. The combination of microscopic rates results in unusually high overall ligand binding rate constants, a fact that contributes to explaining at the mechanistic level the extremely high reactivity of these proteins toward the physiological ligands oxygen, nitric oxide and nitrite.

scholarly journals Qualitative Prediction of Ligand Dissociation Kinetics from Focal Adhesion Kinase Using Steered Molecular Dynamics

Life ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 74
Author(s):  
Justin Spiriti ◽  
Chung F. Wong
Keyword(s):  
Molecular Dynamics ◽  
Drug Discovery ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Early Stage ◽  
Steered Molecular Dynamics ◽  
Drug Binding ◽  
Binding Kinetics ◽  
Dissociation Kinetics ◽  
Binding Characteristics

Most early-stage drug discovery projects focus on equilibrium binding affinity to the target alongside selectivity and other pharmaceutical properties. Since many approved drugs have nonequilibrium binding characteristics, there has been increasing interest in optimizing binding kinetics early in the drug discovery process. As focal adhesion kinase (FAK) is an important drug target, we examine whether steered molecular dynamics (SMD) can be useful for identifying drug candidates with the desired drug-binding kinetics. In simulating the dissociation of 14 ligands from FAK, we find an empirical power–law relationship between the simulated time needed for ligand unbinding and the experimental rate constant for dissociation, with a strong correlation depending on the SMD force used. To improve predictions, we further develop regression models connecting experimental dissociation rate with various structural and energetic quantities derived from the simulations. These models can be used to predict dissociation rates from FAK for related compounds.

Statistical comparison of ligand-binding kinetics

1989 ◽  
Vol 8 (7) ◽  
pp. 871-881
Author(s):  
John B. Cologne ◽  
Paul M. Mendelman ◽  
Donald O. Chaffin
Keyword(s):  
Ligand Binding ◽  
Binding Kinetics ◽  
Statistical Comparison

Fast-phase transvascular fluid flux and the Fahraeus effect

1987 ◽  
Vol 62 (4) ◽  
pp. 1513-1520 ◽  
Author(s):  
W. N. Richardson ◽  
D. Bilan ◽  
M. Hoppensack ◽  
L. Oppenheimer
Keyword(s):  
Mechanical Properties ◽  
Slow Rate ◽  
Time Course ◽  
Rate Of Change ◽  
Distributed Model ◽  
Slow Phase ◽  
Fluid Flux ◽  
Fast Phase ◽  
Constant Rate ◽  
Fahraeus Effect

Transvascular fluid flux was induced in six isolated blood-perfused canine lobes by increasing and decreasing hydrostatic inflow pressure (Pi). Fluid flux was followed against the change in concentration of an impermeable tracer (Blue Dextran) measured directly with a colorimetric device. The time course of fluid flux was biphasic with an initial fast transient followed by a slow phase. Hematocrit changes unrelated to fluid flux occurred due to the Fahraeus effect, and their contribution to the total color signal was subtracted to determine the rate of fast fluid flux (Qf). Qf was related to Pi to derive fast-phase conductance (Kf). Slow-phase Kf was calculated from the constant rate of change of lobe weight. For a mean change in Pi of 7 cmH2O, 40% of the color signal was due to fluid flux. Fast- and slow-phase Kf's were 0.86 +/- 0.15 and 0.27 +/- 0.05 ml X min-1. cmH2O–1 X 100 g dry wt-1. The fast-phase Kf is smaller than that reported for plasma-perfused lobes. Possible explanations discussed are the nature of the perfusate, the mechanical properties of the interstitium, and the slow rate of rise of the driving pressure at the filtration site on the basis of a distributed model of pulmonary vascular compliance.

scholarly journals Roles of cell and microvillus deformation and receptor-ligand binding kinetics in cell rolling

2008 ◽  
Vol 295 (4) ◽  
pp. H1439-H1450 ◽  
Author(s):  
Parag Pawar ◽  
Sameer Jadhav ◽  
Charles D. Eggleton ◽  
Konstantinos Konstantopoulos
Keyword(s):  
Ligand Binding ◽  
Three Dimensional ◽  
Theoretical Models ◽  
Binding Kinetics ◽  
Cell Deformation ◽  
Receptor Ligand ◽  
Length Scales ◽  
Bond Behavior ◽  
Cell Rolling ◽  
Threshold Phenomenon

Polymorphonuclear leukocyte (PMN) recruitment to sites of inflammation is initiated by selectin-mediated PMN tethering and rolling on activated endothelium under flow. Cell rolling is modulated by bulk cell deformation (mesoscale), microvillus deformability (microscale), and receptor-ligand binding kinetics (nanoscale). Selectin-ligand bonds exhibit a catch-slip bond behavior, and their dissociation is governed not only by the force but also by the force history. Whereas previous theoretical models have studied the significance of these three “length scales” in isolation, how their interplay affects cell rolling has yet to be resolved. We therefore developed a three-dimensional computational model that integrates the aforementioned length scales to delineate their relative contributions to PMN rolling. Our simulations predict that the catch-slip bond behavior and to a lesser extent bulk cell deformation are responsible for the shear threshold phenomenon. Cells bearing deformable rather than rigid microvilli roll slower only at high P-selectin site densities and elevated levels of shear (≥400 s−1). The more compliant cells (membrance stiffness = 1.2 dyn/cm) rolled slower than cells with a membrane stiffness of 3.0 dyn/cm at shear rates >50 s−1. In summary, our model demonstrates that cell rolling over a ligand-coated surface is a highly coordinated process characterized by a complex interplay between forces acting on three distinct length scales.

scholarly journals Different SP1 binding dynamics at individual genomic loci in human cells

2020 ◽  
Author(s):  
Yuko Hasegawa ◽  
Kevin Struhl
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Time Course ◽  
Nucleosome Occupancy ◽  
Binding Kinetics ◽  
Common Mechanism ◽  
Sequence Motif ◽  
Promoter Regions ◽  
Pml Bodies ◽  
Slow Turnover

Using a tamoxifen-inducible time-course ChIP-seq approach, we show that the ubiquitous transcription factor SP1 has different binding dynamics at its target sites in the human genome that are not correlated with SP1 occupancy levels at those sites. While ~70% of SP1 binding sites are located in promoter regions, loci with slow SP1 binding turnover are enriched in enhancer and Polycomb-repressed regions. Unexpectedly, SP1 sites with fast turnover times tend to have higher quality and more copies of the SP1 sequence motif. Different co-binding factors associate near SP1 binding sites depending on their binding kinetics and on their location at promoters or enhancers. For example, NFY and FOS are preferentially associated near promoter-bound SP1 sites with fast turnover, whereas DNA motifs of ETS and homeodomain proteins are preferentially observed at sites with slow turnover. At promoters but not enhancers, proteins involved in sumoylation and PML bodies associate more strongly with slow SP1 binding sites than with the fast-binding sites. The speed of SP1 binding turnover is not associated with nucleosome occupancy, and it is not necessarily coupled to higher transcriptional activity. These results with SP1 are in contrast from those of human TBP, indicating that there is no common mechanism affecting transcription factor binding kinetics.

scholarly journals Different SP1 binding dynamics at individual genomic loci in human cells

2021 ◽  
Vol 118 (46) ◽  
pp. e2113579118
Author(s):  
Yuko Hasegawa ◽  
Kevin Struhl
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Time Course ◽  
Nucleosome Occupancy ◽  
Binding Kinetics ◽  
Common Mechanism ◽  
Sequence Motif ◽  
Chip Sequencing ◽  
Target Sites ◽  
Maximal Binding

Using a tamoxifen-inducible time-course ChIP-sequencing (ChIP-seq) approach, we show that the ubiquitous transcription factor SP1 has different binding dynamics at its target sites in the human genome. SP1 very rapidly reaches maximal binding levels at some sites, but binding kinetics at other sites is biphasic, with rapid half-maximal binding followed by a considerably slower increase to maximal binding. While ∼70% of SP1 binding sites are located at promoter regions, loci with slow SP1 binding kinetics are enriched in enhancer and Polycomb-repressed regions. Unexpectedly, SP1 sites with fast binding kinetics tend to have higher quality and more copies of the SP1 sequence motif. Different cobinding factors associate near SP1 binding sites depending on their binding kinetics and on their location at promoters or enhancers. For example, NFY and FOS are preferentially associated near promoter-bound SP1 sites with fast binding kinetics, whereas DNA motifs of ETS and homeodomain proteins are preferentially observed at sites with slow binding kinetics. At promoters but not enhancers, proteins involved in sumoylation and PML bodies associate more strongly with slow SP1 binding sites than with the fast binding sites. The speed of SP1 binding is not associated with nucleosome occupancy, and it is not necessarily coupled to higher transcriptional activity. These results with SP1 are in contrast to those of human TBP, indicating that there is no common mechanism affecting transcription factor binding kinetics. The biphasic kinetics at some SP1 target sites suggest the existence of distinct chromatin states at these loci in different cells within the overall population.

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