Search Results (7)

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (K_d), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC₅₀ approaches the K_d value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to K_d. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols. Full article

The association of two or more proteins to adopt a quaternary complex is one of the most widespread mechanisms by which protein function is modulated. In this scenario, three-dimensional domain swapping (3D-DS) constitutes one plausible pathway for the evolution of protein oligomerization that exploits readily available intramolecular contacts to be established in an intermolecular fashion. However, analysis of the oligomerization kinetics and thermodynamics of most extant 3D-DS proteins shows its dependence on protein unfolding, obscuring the elucidation of the emergence of 3D-DS during evolution, its occurrence under physiological conditions, and its biological relevance. Here, we describe the human FoxP subfamily of transcription factors as a feasible model to study the evolution of 3D-DS, due to their significantly faster dissociation and dimerization kinetics and lower dissociation constants in comparison to most 3D-DS models. Through the biophysical and functional characterization of FoxP proteins, relevant structural aspects highlighting the evolutionary adaptations of these proteins to enable efficient 3D-DS have been ascertained. Most biophysical studies on FoxP suggest that the dynamics of the polypeptide chain are crucial to decrease the energy barrier of 3D-DS, enabling its fast oligomerization under physiological conditions. Moreover, comparison of biophysical parameters between human FoxP proteins in the context of their minute sequence differences suggests differential evolutionary strategies to favor homoassociation and presages the possibility of heteroassociations, with direct impacts in their gene regulation function. Full article

Humira is a monoclonal antibody that binds to TNF alpha, inactivates TNF alpha receptors, and inhibits inflammation. Neonatal Fc receptors can mediate the transcytosis of Humira–TNF alpha complex structures and process them toward degradation pathways, which reduces the therapeutic effect of Humira. Allowing the Humira–TNF alpha complex structures to dissociate to Humira and soluble TNF alpha in the early endosome to enable Humira recycling is crucial. We used the cytoplasmic pH (7.4), the early endosomal pH (6.0), and pK_a of histidine side chains (6.0–6.4) to mutate the residues of complementarity-determining regions with histidine. Our engineered Humira (W1-Humira) can bind to TNF alpha in plasma at neutral pH and dissociate from the TNF alpha in the endosome at acidic pH. We used the constant-pH molecular dynamics, Gaussian accelerated molecular dynamics, two-dimensional potential mean force profiles, and in vitro methods to investigate the characteristics of W1-Humira. Our results revealed that the proposed Humira can bind TNF alpha with pH-dependent affinity in vitro. The W1-Humira was weaker than wild-type Humira at neutral pH in vitro, and our prediction results were close to the in vitro results. Furthermore, our approach displayed a high accuracy in antibody pH-dependent binding characteristics prediction, which may facilitate antibody drug design. Advancements in computational methods and computing power may further aid in addressing the challenges in antibody drug design. Full article

The binding of integrin proteins to peptide sequences such as arginine-glycine-aspartic acid (RGD) is a crucial step in the adhesion process of mammalian cells. While these bonds can be examined between purified proteins and their ligands, live-cell assays are better suited to gain biologically relevant information. Here we apply a computer-controlled micropipette (CCMP) to measure the dissociation constant (K_d) of integrin-RGD-binding. Surface coatings with varying RGD densities were prepared, and the detachment of single cells from these surfaces was measured by applying a local flow inducing hydrodynamic lifting force on the targeted cells in discrete steps. The average behavior of the populations was then fit according to the chemical law of mass action. To verify the resulting value of K_d^2d = (4503 ± 1673) 1/µm², a resonant waveguide grating based biosensor was used, characterizing and fitting the adhesion kinetics of the cell populations. Both methods yielded a K_d within the same range. Furthermore, an analysis of subpopulations was presented, confirming the ability of CCMP to characterize cell adhesion both on single cell and whole population levels. The introduced methodologies offer convenient and automated routes to quantify the adhesivity of living cells before their further processing. Full article

The guanylyl cyclase-activating protein 1, GCAP1, activates or inhibits retinal guanylyl cyclase (retGC) depending on cellular Ca²⁺ concentrations. Several point mutations of GCAP1 have been associated with impaired calcium sensitivity that eventually triggers progressive retinal degeneration. In this work, we demonstrate that the recombinant human protein presents a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and, more importantly, by protein binding to Ca²⁺ or Mg²⁺. Based on small-angle X-ray scattering data, protein-protein docking, and molecular dynamics simulations we propose two novel three-dimensional models of Ca²⁺-bound GCAP1 dimer. The different propensity of human GCAP1 to dimerize suggests structural differences induced by cation binding potentially involved in the regulation of retGC activity. Full article

Thioredoxins (TRXs) are major protein disulfide reductases of the cell. Their redox activity relies on a conserved Trp-Cys-(Gly/Pro)-Pro-Cys active site bearing two cysteine (Cys) residues that can be found either as free thiols (reduced TRXs) or linked together by a disulfide bond (oxidized TRXs) during the catalytic cycle. Their reactivity is crucial for TRX activity, and depends on the active site microenvironment. Here, we solved and compared the 3D structure of reduced and oxidized TRX h1 from Chlamydomonas reinhardtii (CrTRXh1). The three-dimensional structure was also determined for mutants of each active site Cys. Structural alignments of CrTRXh1 with other structurally solved plant TRXs showed a common spatial fold, despite the low sequence identity. Structural analyses of CrTRXh1 revealed that the protein adopts an identical conformation independently from its redox state. Treatment with iodoacetamide (IAM), a Cys alkylating agent, resulted in a rapid and pH-dependent inactivation of CrTRXh1. Starting from fully reduced CrTRXh1, we determined the acid dissociation constant (pK_a) of each active site Cys by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analyses coupled to differential IAM-based alkylation. Based on the diversity of catalytic Cys deprotonation states, the mechanisms and structural features underlying disulfide redox activity are discussed. Full article

Optical biosensors based on one-dimensional photonic crystals sustaining Bloch surface waves are proposed to study antibody interactions and perform affinity studies. The presented approach utilizes two types of different antibodies anchored at the sensitive area of a photonic crystal-based biosensor. Such a strategy allows for creating two or more on-chip regions with different biochemical features as well as studying the binding kinetics of biomolecules in real time. In particular, the proposed detection system shows an estimated limit of detection for the target antibody (anti-human IgG) smaller than 0.19 nM (28 ng/mL), corresponding to a minimum surface mass coverage of 10.3 ng/cm². Moreover, from the binding curves we successfully derived the equilibrium association and dissociation constants (K_A = 7.5 × 10⁷ M⁻¹; K_D = 13.26 nM) of the human IgG–anti-human IgG interaction. Full article