Apparent Kd Research Articles

HOW LIGANDS MODULATE THE GASTRIC H,K-ATPASE ACTIVITY AND ITS INHIBITION BY TEGOPRAZAN.

The introduction of potassium-competitive acid blockers (P-CABs) has been a major innovation in gastric H,K-ATPase inhibition and many laboratories are actively engaged in the development of novel molecules within this class. This work investigates the interaction between H,K-ATPase and tegoprazan, a representative of the P-CABs group, in terms of K+ and H+ binding, through functional and structural analyses. First, by studying the H,K-ATPase activity we found a model to describe the non-Michaelis Menten kinetics through a "ping-pong" mechanism that explains a stoichiometry of 1 H+, 1 K+, and 1 ATP molecule, but also considering the influence of H+ on the ionization states of the protein. A kinetic evaluation of the inhibition of tegoprazan denotes the binding to two different intermediates states with apparent Kd (μM) 0.56 ± 0.04 and 2.70 ± 0.24 at pH 7.2. Molecular dynamics simulations revealed important changes in the interactions of tegoprazan with the transmembrane residues depending on whether the site contains K+ or not. This explains the decrease in affinity as a function of K+ concentration observed in the kinetic experiments. On the other hand, the structures predict that the protonation of tegoprazan is responsible for the change in its dihedral angle. The rotation of the benzimidazole ring allows the inhibitor to be positioned further into the luminal cavity, a situation compatible with the higher inhibition affinity of H,K-ATPase measured at low pH. Results presented herein will provide a basis for the rational design of novel P-CABs ligands.

Dissociation Constant (Kd) Measurement for Small-Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations.

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non-target molecules. Dissociation constant (Kd) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small-molecule targets. The reviewed methods include label-free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore-labeled DNA from nanomaterials. The difference between the measured apparent Kd and the true Kd of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non-binding sequences as controls.

Functionalized PLGA-Based Nanoparticles with Anti-HSV-2 Human Monoclonal Antibody: A Proof of Concept for Early Diagnosis and Targeted Therapy.

Background: Functionalized nanoparticles (NPs) represent a cutting edge in innovative clinical approaches, allowing for the delivery of selected compounds with higher specificity in a wider time frame. They also hold promise for novel theranostic applications that integrate both diagnostic and therapeutic functions. Pathogens are continuously evolving to try to escape the strategies designed to treat them. Objectives: In this work, we describe the development of a biotechnological device, Nano-Immuno-Probes (NIPs), for early detection and infections treatment. Human Herpes Simplex Virus 2 was chosen as model pathogen. Methods: NIPs consist of PLGA-PEG-Sulfone polymeric NPs conjugated to recombinant Fab antibody fragments targeting the viral glycoprotein G2. NIPs synthesis involved multiple steps and was validated through several techniques. Results: DLS analysis indicated an expected size increase with a good polydispersity index. Z-average and z-potential values were measured for PLGA-PEG-Bis-Sulfone NPs (86.6 ± 10.9 nm; -0.7 ± 0.3 mV) and NIPs (151 ± 10.4 nm; -5.1 ± 1.9 mV). SPR assays confirmed NIPs' specificity for the glycoprotein G2, with an apparent KD of 1.03 ± 0.61 µM. NIPs exhibited no cytotoxic effects on VERO cells at 24 and 48 h. Conclusions: This in vitro study showed that NIPs effectively target HSV-2, suggesting the potential use of these nanodevices to deliver both contrast agents as well as therapeutic compounds.

The combination of Asp519Val/Glu665Val and Lys1813Ala mutations in FVIII markedly increases coagulation potential

AbstractA2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by ∼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ∼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ∼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 μg/kg) was equal to that of the WT (2 μg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa.

Specificity and mechanism of TonB-dependent ferric catecholate uptake by Fiu.

We studied the Escherichia coli outer membrane protein Fiu, a presumed transporter of monomeric ferric catecholates, by introducing Cys residues in its surface loops and modifying them with fluorescein maleimide (FM). Fiu-FM bound iron complexes of the tricatecholate siderophore enterobactin (FeEnt) and glucosylated enterobactin (FeGEnt), their dicatecholate degradation product Fe(DHBS)2 (FeEnt*), the monocatecholates dihydroxybenzoic acid (FeDHBA) and dihydroxybenzoyl serine (FeDHBS), and the siderophore antibiotics cefiderocol (FDC) and MB-1. Unlike high-affinity ligand-gated porins (LGPs), Fiu-FM had only micromolar affinity for iron complexes. Its apparent KD values for FeDHBS, FeDHBA, FeEnt*, FeEnt, FeGEnt, FeFDC, and FeMB-1 were 0.1, 0.7, 0.7, 1.0, 0.3, 0.4, and 4 μM, respectively. Despite its broad binding abilities, the transport repertoires of E. coli Fiu, as well as those of Cir and FepA, were less broad. Fiu only transported FeEnt*. Cir transported FeEnt* and FeDHBS (weakly); FepA transported FeEnt, FeEnt*, and FeDHBA. Both Cir and FepA bound FeGEnt, albeit with lower affinity. Related transporters of Acinetobacter baumannii (PiuA, PirA, BauA) had similarly moderate affinity and broad specificity for di- or monomeric ferric catecholates. Both microbiological and radioisotopic experiments showed Fiu's exclusive transport of FeEnt*, rather than ferric monocatecholate compounds. Molecular docking and molecular dynamics simulations predicted three binding sites for FeEnt*in the external vestibule of Fiu, and a fourth site deeper in its interior. Alanine scanning mutagenesis in the outermost sites (1a, 1b, and 2) decreased FeEnt* binding affinity as much as 20-fold and reduced or eliminated FeEnt* uptake. Finally, the molecular dynamics simulations suggested a pathway of FeEnt* movement through Fiu that may generally describe the process of metal transport by TonB-dependent receptors.

Engineering a monobody specific to monomeric Cu/Zn-superoxide dismutase associated with amyotrophic lateral sclerosis.

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies. Here, we have developed Mb(S4) monobody, a small synthetic binding protein based on the fibronectin type III scaffold, that recognizes a monomeric but not dimeric form of SOD1 by performing combinatorial library selections using phage and yeast-surface display methods. Although Mb(S4) was characterized by its excellent selectivity to the monomeric conformation of SOD1, the monomeric SOD1/Mb(S4) complex was not so stable (apparent Kd ~ μM) as to be detected in conventional pull-down experiments. Instead, the complex of Mb(S4) with monomeric but not dimeric SOD1 was successfully trapped by proximity-enabled chemical crosslinking even when reacted in the cell lysates. We thus anticipate that Mb(S4) binding followed by chemical crosslinking would be a useful strategy for in vitro and also ex vivo detection of the monomeric SOD1 proteins.

Sorption behavior of cesium ions to Mg-containing calcium silicate hydrate in a co-precipitation process

The sorption behavior of Cs to Mg-containing calcium silicate hydrate (C–S–H) was examined to consider C–S–H formation under the water-saturated conditions around a radioactive waste repository. The amount of Cs sorption was slightly lower for Mg-containing C–S–H than for C–S–H, because Mg leads to a decrease in sorption sites by facilitating the polymerization of silicate chains. However, the apparent sorption distribution coefficient Kd of Cs on C–S–H with a Ca/Si molar ratio of 0.8 and Mg contents up to 20% was estimated to be 3.4–6.1 mL/g. Furthermore, the amount of Cs sorption was not decreased by the presence of Ba. The apparent Kd obtained in this study was about 10 times higher than that conventionally estimated for host rocks in the plutonic repository, indicating that C–S–H retains the effect of Cs immobilization even when it contains Mg.Graphical abstract

Interaction of full-length Tau with negatively charged lipid membranes leads to polymorphic aggregates.

The Tau protein is implicated in various diseases collectively known as tauopathies, including Alzheimer's disease and frontotemporal dementia. The precise mechanism underlying Tau pathogenicity remains elusive. Recently, the role of lipids has garnered interest due to their implications in Tau aggregation, secretion, uptake, and pathogenic dysregulation. Previous investigations have highlighted critical aspects: (i) Tau's tendency to aggregate into fibers when interacting with negatively charged lipids, (ii) its ability to form structured species upon contact with anionic membranes, and (iii) the potential disruption of the membrane upon Tau binding. In this study, we examine the disease-associated P301L mutation of the 2N4R isoform of Tau and its effects on membranes composed on phosphatidylserine (PS) lipids. Aggregation studies and liposome leakage assays demonstrate Tau's ability to bind to anionic lipid vesicles, leading to membrane disruption. Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) reveals the accumulation of Tau on the membrane surface without protein insertion, structuration, or lipid removal. Plasmon waveguide resonance (PWR) demonstrates a strong binding of Tau on PS bilayers with an apparent Kd in the micromolar range, indicating the deposition of a thick protein layer. Atomic force microscopy (AFM) real-time imaging allows the observation of partial lipid solubilization and the deposition of polymorphic aggregates in the form of thick patches and fibrillary structures resembling amyloid fibers, which could grow from a combination of extracted anionic phospholipids from the membrane and Tau protein. This study deepens our understanding of full-length Tau's multifaceted interactions with lipids, shedding light on potential mechanisms leading to the formation of pathogenic Tau assemblies.

Competitive aptamer switch for modulating ligand binding affinity

Aptamers are short, single-stranded DNA or RNA molecules that selectively bind to a target molecule. Aptamer-complement duplex (ACD) is often used to design molecular switches capable of producing a detectable signal or triggering a structural change upon aptamer binding to a target. However, such aptamer switch generally faces an increased dissociation constant (Kd) due to the energy barrier of the complementary duplex. We reported a competitive hybridization mechanism to modulate the binding affinity of an ACD to a target adenosine. Using the computation-guided design, we calculated the aptamer folding energy for the duplex length from 11-nt to 15-nt, and experimentally measured increased apparent Kd values resulted from these extended duplexes. Using a set of strands to compete with the ACD hybridization, it reduced the aptamer folding energy to facilitate aptamer switches with decreased apparent Kd values ranging from over 400 μM without a competing strand to ∼30 μM with a competing strand. This competitive aptamer switch was also found sensitive to single-nucleotide mutations of a competing strand. Our work provides an approach to modulate the binding affinity and the sensitivity of aptamer-complement duplexes, which could be useful in the nucleic acids-based sensing and nanomedicine.

A Novel Anti-CD44 Variant 3 Monoclonal Antibody C44Mab-6 Was Established for Multiple Applications.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3-10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3-10) or some cancer cell lines (COLO205 and HSC-3) via flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3-10, COLO205, and HSC-3 was 1.5 × 10-9 M, 6.3 × 10-9 M, and 1.9 × 10-9 M, respectively. C44Mab-6 could detect the CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments and is expected for the application of tumor diagnosis and therapy.

Development of a Novel Anti-CD44 Variant 5 Monoclonal Antibody C44Mab-3 for Multiple Applications against Pancreatic Carcinomas.

Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker and plays tumor promotion and drug resistance roles in various cancers. In particular, the splicing variants are overexpressed in many carcinomas and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant's (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant-5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3-10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3-10 and PK-1 was 1.3 × 10-9 M and 2.6 × 10-9 M, respectively. C44Mab-3 could detect the exogenous CD44v3-10 and endogenous CD44v5 in Western blotting and stained the formalin-fixed paraffin-embedded pancreatic cancer cells but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic cancer diagnosis and therapy.

Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10-9 and 3.2 × 10-9 M, respectively. C44Mab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.

Characterization of the Japanese Quail Oocyte Receptor for Very Low Density Lipoprotein and Vitellogenin

The plasma membrane receptor for VLDL and vitellogenin from oocytes of Japanese quail (Coturnix coturnix japonica) was characterized and compared with that of another domestic fowl, the chicken (Gallus domesticus). When visualized by ligand blotting with biotinylated or 125I-labeled lipoproteins, the quail VLDL/vitellogenin receptor had an apparent Mr of 95 kDa under nonreducing conditions, identical to that of the chicken receptor. Upon analysis by ligand blotting, binding of radiolabeled quail plasma VLDL to the quail oocyte receptor seemed to be saturable and exhibited high affinity (apparent Kd of 13.9 mg/L). Cross-reactivity, at the level of ligand recognition, was observed between quail and chicken VLDL/vitellogenin receptors, and immunological relatedness was demonstrated by Western blotting with a rabbit anti-chicken oocyte VLDL receptor antibody. In contrast, a species difference was observed in the apolipoprotein VLDL-II moiety of plasma VLDL. Chicken apolipoprotein VLDL-II, an 82-amino acid protein with a disulfide crosslink at residue 75 (the sole cysteine residue), existed as a homodimer of 9.5 kDa subunits and, to a lesser extent, as a monomer. Quail apolipoprotein VLDL-II existed only in monomeric form without reduction and lacked cysteine. The present results demonstrate that, despite a difference in an apolipoprotein moiety of VLDL, quail and chicken oocyte lipoprotein receptors share key structural and functional elements. This lends further support to the notion that receptor recognition is mediated by the common VLDL component, apolipoprotein B.

7S Globulin from Soybean Is Metabolized in Human Cell Cultures by a Specific Uptake and Degradation System

We examined the biological fate of 7S globulin from soybean in a hepatoma cell line (Hep G2) and in human skin fibroblasts (HSF) to gain new insights into the 7S globulin cell process, the final effect of which is an enhanced expression of the LDL-receptor. The ability of 7S globulin to bind and to be internalized and degraded by both cell types was investigated under different experimental conditions. In all cases, specific uptake (binding + internalization) and degradation of 125I-7S globulin were curvilinear functions of substrate concentration at 37°C. The two processes were saturated at around 80 mg/L, a concentration at which an up-regulation of LDL-receptor was previously reported. The specific uptake of 125I-7S globulin at 37°C was a curvilinear function of time, and achieved equilibrium after 6 and 12 h in HSF and Hep G2 cells, respectively. Binding experiments, conducted at 4°C in Hep G2 cells, showed a specific and saturable association of 7S globulin to the cell membrane. Linear Scatchard analysis demonstrated a single population of binding sites. The amount of 7S globulin bound at saturation (Bmax) was about 2.73 mg/L, with an apparent Kd of 21 µmol/L, assuming 175 kDa as the 7S globulin molecular weight. SDS-PAGE of Hep G2 membrane proteins incubated with 125I-7S globulin revealed a specific interaction of 7S globulin with a cell protein component with molecular weight between 14 and 21 kDa. Further studies are needed to ascertain whether this interaction is directly or indirectly related to the observed stimulation of the LDL-receptor.

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation.

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KD app for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl- on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl-), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208s-1 for D-glucose to 95s-1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,K app = 210mM). Affinity for D-glucose increases 14-fold (KD,Na app = 15mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9mM) compared to KD,Na app due to optimized kinetics. In contrast, KM and KD app values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.

Calmodulin binds FGF12A to regulate Nav1.5 channel inactivation.

Activation and inactivation gating of the Nav1.5 voltage-gated ion channel is responsible for the rapid depolarization of the cardiac action potential (AP) and regulates its duration. Altered inactivation kinetics can lead to AP prolongation and potentiate arrhythmia. Th Nav1.5 C-terminal domain (CTD) is known to modulate inactivation. In addition, there are multiple accessory proteins that bind the CTD are CaM and FGF12A. CaM has been linked to a possible Ca2+ modulation of the Nav1.5 as Ca2+ ions typically do not interact directly with the channel. However, the combined interaction on channel inactivation between CaM and FGF12A has also not been determined. We employed the use of cut-open voltage clamping to record the current traces. FRET imaging was used to assess binding affinity between FGF12A and CaM with apparent KD values. The addition of FGF12A to the CTD induces shifts in the activation's voltage dependence (+10.0 ±1.9 SEM mV, p<0.05) and removing CaM with the IQ/AA mutation on Nav1.5 leads to a depolarizing shift in inactivation (+19.5 ±2.1 SEM mV, p<0.01). We have shown that the addition of FGF12A to a channel lacking CaM (IQ/AA) will have a depolarizing shift in the steady state inactivation (+15.5 ±2.6 SEM mV, p<0.01). Furthermore, we have shown that a binding domain for CaM on the FGF12A protein itself requires the presence of Ca2+ ions to associate by measuring increase in the apparent KD value determined through FRET imaging. By removing this CaM binding domain from FGF12A we see a reduction in the recovery from inactivation and a hyperpolarizing shift in the voltage dependence of inactivaiton (−10.0 ±2.8 mV SEM, p<0.0005). The shift in inactivation based upon the presence of CaM could indicate an interaction between Ca2+ ions and the Nav1.5 channel.

Reductionist insights into how chaperone charge influences RNA folding.

Non-coding RNAs are composed of modular secondary and tertiary structural units that themselves must form complex 3D structures for the entire RNA to fold and perform its biological function. However, the high density of negative charges associated with the phosphate backbone gives rise to substantial amounts of electrostatic repulsion, contributing to the large energetic barrier between unfolded and folded states. RNA folding chaperones (RFCs) are proteins that help lower this energy barrier by transiently interacting with RNA. Recent work has shown that some RFCs can function as macromolecular counterions for their RNA clients, thereby screening backbone electrostatic repulsion. However, the fundamental principles associated with how RFCs assist folding is largely unexplored. Here, we reduce RFCs to their smallest cationic constituents and then systematically “re-build” RFCs from these components (e.g., NH4+→ Lys→ polyK10→ RFC). We use single-molecule FRET to determine how these counterions influence both the relative population abundance and mean FRET efficiency of the folded and unfolded states of small model RNAs. From these studies, we have begun to determine aspects of RFCs that contribute to chaperoning RNA folding, such as net cationic charge and charge distribution. Here, we show that cationic amino acids (e.g., Lys and Arg) bind to RNA with apparent Kd values (appKd) on the order of 100 mM, which are comparable to those associated with their characteristic functional groups (e.g., ammonium and guanidinium). Further, increasing the net cationic charge of a counterion systematically decreases the appKd over several orders of magnitude. We also show a correlation between the decrease in appKd and a decrease in the folding free energy barrier. We then compare our observations of these polypeptide chaperones to the behavior of known RFCs (e.g., protamine, H1, and HCV nucleocapsid protein) with the small model RNAs.

Quantifying membrane binding and diffusion with fluorescence correlation spectroscopy diffusion laws

Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g., single/multiple particle tracking, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy (FCS), enable non-invasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non-Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant KD, the diffusion coefficient on the membrane D2D, and the diffusion coefficient in the cytosol, D3D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells, and we quantified KD and D2D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (KD and D2D) for peripheral membrane molecules.

Impact of human serum albumin on CuII and ZnII complexation by ATSM (diacetyl-bis(N4-methylthiosemicarbazone)) and a water soluble analogue.

The chelator diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) and its complexes with CuII and ZnII are becoming increasingly investigated for medical applications such as PET imaging for anti-tumour therapy and the treatment of amyotrophic lateral sclerosis. However, the solubility in water of both the ligand and the complexes presents certain limitations for in vitro studies. Moreover, the stability of the CuII and ZnII complexes and their metal exchange reaction against the potential biological competitor human serum albumin (HSA) has not been studied in depth. In this work it was observed that the ATSM with an added carboxylic group into the structure increases its solubility in aqueous solutions without altering the coordination mode and the conjugated system of the ligand. The poorly water-soluble CuII- and ZnII-ATSM complexes were prevented from precipitating due to the binding to HSA. Both HSA and ATSM show a similar thermodynamic affinity for ZnII. Finally, the CuII-competition experiments with EDTA and the water-soluble ATSM ligands yielded an apparent log Kd at pH 7.4 of about -19. When ATSM was added to CuII- and ZnII-loaded HSA, withdrawing of ZnII was kinetically favoured, but this metal is slowly substituted by the CuII afterwards taken from HSA so that this protein could be considered as a source of CuII for ATSM.

Fluorometric biosensing of α-amylase using an artificial allosteric biosensor immobilized on nanostructured interface

Protein biosensors hold a promise to transform the way we collect physiological data by enabling quantification of biomarkers outside of specialized laboratory environment. However, achieving high specificity and sensitivity in homogeneous assay format remains challenging. Here we report construction of fluorescent biosensor arrays based on artificial allosteric α-amylase-activated PQQ-dependent glucose dehydrogenase (Amy-GDH). Amy-GDH was covalently immobilized on silica nanoparticles that were then arrayed on fiberglass sheets. The activity of the biosensor was monitored using a smartphone camera via emergence of bright fluorescence (λex 365 nm) originating from reduced phenazine methosulfate upon glucose oxidation by Amy-GDH. We show that such biosensor arrays demonstrate an apparent Kd of 115 pM for α-amylase with a detection limit of 2 pM. Using the developed biosensor arrays, we were able to specifically and accurately quantify the concentration of α-amylase in biological fluids such as serum and saliva. We propose that the presented approach can enable construction of ultrasensitive point-of-care diagnostic arrays.