skip to main content

This content will become publicly available on September 1, 2024

Title: Determination of binding constants by ultrafast affinity extraction: Theoretical and experimental studies of optimum conditions for analysis
Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α1-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F0 ≈ 0.20-0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents.  more » « less
Award ID(s):
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Date Published:
Journal Name:
Journal of Chromatography A
Page Range / eLocation ID:
Subject(s) / Keyword(s):
["Ultrafast affinity extraction","Drug-protein binding","Free drug fraction","Association equilibrium constant","Affinity microcolumn"]
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. AQP7 is one of the four human aquaglyceroporins that facilitate glycerol transport across the cell membrane, a biophysical process that is essential in human physiology. Therefore, it is interesting to compute AQP7's affinity for its substrate (glycerol) with reasonable certainty to compare with the experimental data suggesting high affinity in contrast with most computational studies predicting low affinity. In this study aimed at computing the AQP7-glycerol affinity with high confidence, we implemented a direct computation of the affinity from unbiased equilibrium molecular dynamics (MD) simulations of three all-atom systems constituted with 0.16 M, 4.32 M, and 10.23 M atoms, respectively. These three sets of simulations manifested a fundamental physics law that the intrinsic fluctuations of pressure in a system are inversely proportional to the system size (the number of atoms in it). These simulations showed that the computed values of glycerol-AQP7 affinity are dependent upon the system size (the inverse affinity estimations were, respectively, 47.3 mM, 1.6 mM, and 0.92 mM for the three model systems). In this, we obtained a lower bound for the AQP7-glycerol affinity (an upper bound for the dissociation constant). Namely, the AQP7-glycerol affinity is stronger than 1087/M (the dissociation constant is less than 0.92 mM). Additionally, we conducted hyper steered MD (hSMD) simulations to map out the Gibbs free-energy profile. From the free-energy profile, we produced an independent computation of the AQP7-glycerol dissociation constant being approximately 0.18 mM. 
    more » « less
  2. Dibenzo-18-crown-6 (DB18C6) is a single-crown ether that can act as a host for a guest ion. In an effort to illuminate the relationships among structure, dynamics, and thermodynamics of ligand binding in a simple model for understanding the affinity and specificity of ligand interactions, nuclear magnetic resonance (NMR) experiments and density functional theory (DFT) were used to study the interaction of DB18C6 with ammonium ion. 1H-NMR was used to follow the titration of DB18C6 with ammonium chloride in deuterated methanol, a solvent chosen for its amphipathic character. Ammonium ion binds strongly to DB18C6 with a dissociation equilibrium constant at least as low as ~ 10 - 6 M. DFT calculations were used to identify optimized conformations of bound and free DB18C6 and to estimate its binding energy with ammonium ion in implicit solvent. An approach is described that accounts for geometry relaxation in addition to solvation correction and basis set superposition error; to our knowledge, this is the first such report that includes the energy difference from optimizing species geometry. The lowest-energy conformer of free DB18C6 in implicit methanol acquires an open, W-shaped structure that is also the lowest-energy conformer found for the DB18C6-ammonium ion complex. These results form a foundation for further studies of this system by molecular dynamics simulations. 
    more » « less
  3. null (Ed.)
    We present a quantitative study comparing the binding of 4-methoxypyridine, MeOPy, ligand to Co( ii )octaethylporphyrin, CoOEP, at the phenyloctane/HOPG interface and in toluene solution. Scanning tunneling microscopy (STM) was used to study the ligand binding to the porphyrin receptors adsorbed on graphite. Electronic spectroscopy was employed for examining this process in fluid solution. The on surface coordination reaction was completely reversible and followed a simple Langmuir adsorption isotherm. Ligand affinities (or Δ G ) for the binding processes in the two different chemical environments were determined from the respective equilibrium constants. The free energy value of −13.0 ± 0.3 kJ mol −1 for the ligation reaction of MeOPy to CoOEP at the solution/HOPG interface is less negative than the Δ G for cobalt porphyrin complexed to the ligand in solution, −16.8 ± 0.2 kJ mol −1 . This result indicates that the MeOPy–CoOEP complex is more stable in solution than on the surface. Additional thermodynamic values for the formation of the surface ligated species (Δ H c = −50 kJ mol −1 and Δ S c = −120 J mol −1 ) were extracted from temperature dependent STM measurements. Density functional computational methods were also employed to explore the energetics of both the solution and surface reactions. At high concentrations of MeOPy the monolayer was observed to be stripped from the surface. Computational results indicate that this is not because of a reduction in adsorption energy of the MeOPy–CoOEP complex. Nearest neighbor analysis of the MeOPy–CoOEP in the STM images revealed positive cooperative ligand binding behavior. Our studies bring new insights to the general principles of affinity and cooperativity in the ligand–receptor interactions at the solution/solid interface. Future applications of STM will pave the way for new strategies designing highly functional multisite receptor systems for sensing, catalysis, and pharmacological applications. 
    more » « less
  4. Abstract

    Rapid discovery and development of serum-stable, selective, and high affinity peptide-based binders to protein targets are challenging. Angiotensin converting enzyme 2 (ACE2) has recently been identified as a cardiovascular disease biomarker and the primary receptor utilized by the severe acute respiratory syndrome coronavirus 2. In this study, we report the discovery of high affinity peptidomimetic binders to ACE2 via affinity selection-mass spectrometry (AS-MS). Multiple high affinity ACE2-binding peptides (ABP) were identified by selection from canonical and noncanonical peptidomimetic libraries containing 200 million members (dissociation constant,KD = 19–123 nM). The most potent noncanonical ACE2 peptide binder, ABP N1 (KD = 19 nM), showed enhanced serum stability in comparison with the most potent canonical binder, ABP C7 (KD = 26 nM). Picomolar to low nanomolar ACE2 concentrations in human serum were detected selectively using ABP N1 in an enzyme-linked immunosorbent assay. The discovery of serum-stable noncanonical peptidomimetics like ABP N1 from a single-pass selection demonstrates the utility of advanced AS-MS for accelerated development of affinity reagents to protein targets.

    more » « less
  5. D’Auria, Sabato (Ed.)

    The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs.

    more » « less