Influence of Precise Polymer Conjugation on Aptamer-Target Binding.

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.

Molecular probes for imaging tissue remodeling

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc gamma receptors (FcgammaRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcgammaR interactions are hence important parameters for understanding FcgammaR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcgammaRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 x 10(3), 8.2 x 10(3), 1.1 x 10(4) and 1.8 x 10(4) m(-1) s(-1), off-rates of 4.7 x 10(-3), 5.7 x 10(-3), 5.9 x 10(-3), and 1.9 x 10(-2) s(-1), and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 mum, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcgammaR interactions.

Interaction of Ribonuclease III with the Regulatory Macrodomain Protein YmdB Analyzed by Docking Calculations and SPR Experiments

This paper reports the previously unknown interactions between eight low molecular weight commercially available drugs (130-800 Da) and DNA repair protein photolyase using computational docking simulations and surface plasmon resonance (SPR) experiments. Theoretical dissociation constants, K(d), obtained from molecular docking simulations were compared with the values found from SPR experiments. Among the eight drugs analyzed, computational and experimental values showed similar binding affinities between selected drug and protein pairs. We found no significant differences in binding interactions between pure and commercial forms of the drug lornoxicam and DNA photolyase. Among the eight drugs studied, prednisone, desloratadine, and azelastine exhibited the highest binding affinity (K(d) = 1.65, 2.05, and 8.47 μM, respectively) toward DNA photolyase. Results obtained in this study are promising for use in the prediction of unknown interactions of common drugs with specific proteins such as human clock protein cryptochrome.

Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.

High-Affinity Small Molecule Inhibitors of T Cell Costimulation: Compounds for Immunotherapy

A single-step, high throughput, and highly reproducible method for measuring IgM quantity and avidity directly from fish serum via biolayer interferometry (BLI)

SATB1 is a transcriptional regulator controlling the gene expression that is essential in the maturation of the immune T-cell. SATB1 binds to the nuclear matrix attachment regions of DNA, where it recruits histone deacetylase and represses transcription through a local chromatin remodeling. Here we determined the solution structure of the matrix attachment region-binding domain, possessing similarity to the CUT DNA-binding domain, of human SATB1 by NMR spectroscopy. The structure consists of five alpha-helices, in which the N-terminal four are arranged similarly to the four-helix structure of the CUT domain of hepatocyte nuclear factor 6alpha. By an NMR chemical shift perturbation analysis and by surface plasmon resonance analyses of SATB1 mutant proteins, an interface for DNA binding was revealed to be located at the third helix and the surrounding regions. Surface plasmon resonance experiments using groove-specific binding drugs and methylated DNAs indicated that the domain recognizes DNA from the major groove side. These observations suggested that SATB1 possesses a DNA-binding mode similar to that of the POU-specific DNA-binding domain, which is known to share structural similarity to the four-helix CUT domain.

Difference between follistatin isoforms in the inhibition of activin signalling:: Activin neutralizing activity of follistatin isoforms is dependent on their affinity for activin

Pharmaco-toxicological effects of the synthetic cannabinoids 4F-ABUTINACA, SDB-005, and JWH-018 in mice. In vitro and in vivo studies.

An antitumor fungal polysaccharide from Fomitopsis officinalis by activating immunity and inhibiting angiogenesis

Interaction of an esophageal MEG protein from schistosomes with a human S100 protein involved in inflammatory response

Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies

Artemisia keiskeana Miq. alleviates oxidative stress and ferroptosis in APAP-induced liver injury by mediating Nrf2/GPX4/NF-κB signaling pathway through ESR1.