scholarly journals Association of Model Neurotransmitters with Lipid Bilayer Membranes

2019 ◽  
Author(s):  
B. Josey ◽  
F. Heinrich ◽  
V. Silin ◽  
M. Lösche
Keyword(s):  
Surface Plasmon Resonance ◽  
Ion Channels ◽  
Lipid Bilayer ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Dissociation Constants ◽  
Measurement Techniques ◽  
Neutron Reflectometry ◽  
Membrane Association ◽  
Lipid Bilayer Membranes

AbstractAimed to reproduce the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neuro-transmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neuro-transmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflectometry on artificial membranes reveals the structural aspects of neurotransmitters association with zwitterionic and anionic bilayers. We establish that serotonin interacts non-specifically with the membrane at physiologically relevant concentrations whilst GABA (γ-aminobutyric acid) does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity and neutron reflectometry shows that it penetrates the membrane deeply whereas GABA is excluded from the bilayer.SignificanceReceptor ion channels in the postsynaptic membrane and their neurotransmitter agonists enable fast communication between neuronal cells. Electrophysiology studies reveal surprisingly complex kinetics that apparently require a variety of protein conformational states for their quantitative interpretation, but an alternate hypothesis invoking neurotransmitter membrane association reduces the complexity of the underlying reaction schemes significantly. While their affinity may be low, and is hard to quantify experimentally, neurotransmitter membrane association can be relevant because of their large temporary concentration in the synaptic cleft. With thermodynamic and structural measurements we quantify membrane-bound states of serotonin, establishing this neurotransmitter as membrane-affine, whereas the affinity of the more hydrophilic GABA is too low to register in our sensitivity-optimized measurement techniques.

scholarly journals High-Affinity, Protective Antibodies to the Binding Domain of Botulinum Neurotoxin Type A

2001 ◽  
Vol 69 (1) ◽  
pp. 570-574 ◽  
Author(s):  
Dorothy D. Pless ◽  
Edna R. Torres ◽  
Emily K. Reinke ◽  
Sina Bavari
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Botulinum Neurotoxin ◽  
Epitope Mapping ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Domain ◽  
Protective Antibodies ◽  
Fusion Products

ABSTRACT Monoclonal antibodies (MAbs) were prepared against the putative binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa fragment. Initially, all fusion products were screened against the holotoxin BoNT/A and against the binding fragment, BoNT/A HC. Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A HC was demonstrated by surface plasmon resonance, with dissociation constants ranging from 0.9 to <0.06 nM. Epitope mapping by real-time surface plasmon resonance showed that the antibodies bound to at least two distinct regions of the BoNT/A HC fragment. These MAbs will be useful tools for studying BoNT/A interactions with its receptor, and they have potential diagnostic and therapeutic applications.

Purification of Anti-HPA-1a Directly from Plasma Using Immobilised Recombinant HPA-1a Enables Determination of Antibody Affinity by Surface Plasmon Resonance Technology in Neonatal Alloimmune Thrombocytopenia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3402-3402 ◽  
Author(s):  
Hagop Bessos ◽  
Pamela Brown ◽  
Gholamreza Anani Sarab ◽  
Shirley Gibson ◽  
Michael Moss ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Dissociation Constants ◽  
Plasma Samples ◽  
Antibody Affinity ◽  
High Background ◽  
Alloimmune Thrombocytopenia ◽  
Sepharose 4B

Abstract Background: Most severe cases of neonatal alloimmune thrombocytopenia (NAIT) in the Caucasian population are due to anti-HPA-1a. The precise influences of antibody amount or affinity on severity of NAIT remain to be elucidated. In an earlier study, we assessed the interaction between anti-HPA-1a and HPA-1a antigen by surface plasmon resonance technology (SPRT) using IgG fractions recovered from antibody (ab) positive HPA-1b maternal sera using protein-A chromatography. However, high background in some of the IgG fractions prohibited the determination of antibody affinity (Bessos et al, Trans Apher Sci, In Press). The aim of this study was to investigate the affinity of anti-HPA-1a purified directly from plasma using immobilised HPA-1a antigen. Methods: Native glycoprotein (GP) IIb/IIIa was extracted and purified from HPA-1a1a platelet donors, while recombinant PSI domain of HPA-1a GPIIIa (rec-HPA-1a) was obtained by conventional cloning techniques. CNBr activated Sepharose 4B was coupled either with native GPIIb/IIIa or with rec-HPA-1a and used for the immunopurification of anti-HPA-1a directly from 4 female plasma samples positive for anti-HPA-1a ab. The plasma samples were pre-adsorbed with non-coupled Sepharose 4B prior to mixing with immobilised HPA-1a antigen. The acid eluted abs were dialysed, concentrated and assessed by ELISA (using HPA-1a GPIIb/IIIa coated plates), silver staining (using reduced gels) and SPRT-Biacore X, using CM5 sensor chips bound with HPA-1a1a or HPA-1b1b antigen at 600–800 response units (RU). GPIIIa AP3 monoclonal ab (Mab) & CamTran 007 recombinant anti-HPA-1a (r-anti-1a) were used as controls in SPRT. Affinity and dissociation constants (Ka and Kd respectively) of abs tested in serial doubling dilutions were determined using the computer software BIAevaluation 3.2 RC1 (Biacore) - Langmuir 1:1 mathematical model. Results: Control AP3 Mab bound to both HPA-1a & -1b chips in SPRT yielding respective Ka values of 2x105 & 1.7x104, and respective Kd values of 2.6x10−3 & 6.7x10−5; while control r-anti-1a bound specifically to HPA-1a chips yielding Ka and Kd of 2.9x105 & 8.3x10−4. Antibodies purified from plasma using immobilised rec-HPA-1a demonstrated higher binding in the ELISA and purer samples by silver staining (IgG heavy and light chains) compared to abs purified using immobilised native GP. This was reflected in SPRT where anti-HPA-1a purified using immobilised native GP exhibited high background that prohibited Ka and Kd determinations, whereas abs purified using immobilised rec-HPA- 1a bound specifically to HPA-1a chips, enabling Ka and Kd determinations which ranged from 6.9x103 to 7.9x104 for Ka, and from 1.3x10−4 to 3.4x10−4 for Kd. Anti-HPA-1a purified by immobilised rec-HPA-1a also interacted specifically with CM 5 sensor chips bound with rec-HPA-1a but not rec-HPA-1b, although with lower affinity constants compared to chips bound with native HPA-1a GP, yielding Ka of 4.2x102 to 9.1x103, & Kd of 3.4x10−3 to 8.6x10−5. Conclusion: This is the first report of the direct purification of anti-HPA-1a from plasma by immobilised HPA-1a antigen. Our study shows that immunopurification of plasma using immobilised rec-HPA-1a yields pure anti-HPA-1a antibodies that bind specifically in SPRT to chips coupled with both native or recombinant HPA-1a, allowing determination of antibody affinity and dissociation constants. This novel development would enable much needed analysis of anti-HPA-1a binding mechanisms in NAIT involving patients with different disease outcomes.

scholarly journals Kinetic Analysis and Epitope Mapping of Monoclonal Antibodies to Salmonella Typhimurium Flagellin Using a Surface Plasmon Resonance Biosensor

Antibodies ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 22 ◽  
Author(s):  
Devendra Bhandari ◽  
Fur-Chi Chen ◽  
Shreya Hamal ◽  
Roger Bridgman
Keyword(s):  
Monoclonal Antibodies ◽  
Surface Plasmon Resonance ◽  
Salmonella Typhimurium ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Epitope Mapping ◽  
Dissociation Constants ◽  
Interaction Model ◽  
Dissociation Rate ◽  
Surface Plasmon Resonance Biosensor

Salmonella Typhimurium is one of the leading causes of foodborne diseases worldwide. Biosensors and immunoassays utilizing monoclonal antibodies are widely used for the detection and subtyping of S. Typhimurium. However, due to insufficient information on the nature of binding with S. Typhimurium flagellin, the selection of appropriate antibodies for assay development is a cumbersome task. Hence, we aimed to compare the binding kinetics of a panel of monoclonal antibodies and their relative binding sites to flagellin antigen using a surface plasmon resonance biosensor. Initially, the flagellin was captured on the sensor surface through an immobilized anti-flagellin antibody. The interactions of different concentrations of monoclonal antibodies to flagellin were determined, and binding curves were fitted using 1:1 bio-interaction model to calculate the kinetic parameters. For epitope mapping, pairwise comparisons were completed to determine the binding inhibition of each paired combination of monoclonal antibodies. It was found that these monoclonal antibodies differed significantly (p < 0.05) in association rate, dissociation rate, and equilibrium dissociation constants. Of the five monoclonal antibodies, only two interfered with the binding of each other. Four distinct epitopes located within a 23 kDa domain of flagellin were identified. Findings from this study provide crucial information needed for the further development and optimization of biosensors and other immunoassays for the detection and subtyping of Salmonella.

Surface Plasmon Resonance Study of G Protein/Receptor Coupling in a Lipid Bilayer-Free System

2006 ◽  
Vol 78 (4) ◽  
pp. 1228-1234 ◽  
Author(s):  
Konstantin E. Komolov ◽  
Ivan I. Senin ◽  
Pavel P. Philippov ◽  
Karl-Wilhelm Koch
Keyword(s):  
Surface Plasmon Resonance ◽  
G Protein ◽  
Lipid Bilayer ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Free System ◽  
Receptor Coupling ◽  
Protein Receptor

Affinity analysis between trypsin and aptamers using surface plasmon resonance competition experiments in a steady state

2019 ◽  
Vol 11 (24) ◽  
pp. 3061-3065 ◽  
Author(s):  
Yun Fa ◽  
Mingyang Guan ◽  
Haijie Zhao ◽  
Fei Li ◽  
Huizhou Liu
Keyword(s):  
Surface Plasmon Resonance ◽  
Steady State ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Dissociation Constants ◽  
Competition Experiment ◽  
Dna Aptamers

A surface plasmon resonance (SPR) competition experiment in a steady state was developed to determine the binding dissociation constants between a protein and its DNA aptamers.

Surface Plasmon Resonance Study of Apoptotic Regulators Bcl-2, Bcl-w, Bcl-XL, and Mcl-1 Indicate That the Preclinical Small Molecule Inhibitor (SMI) TW-37 Binds to the Hydrophobic Groove Competitively with tBid To Form a Heterodimer Which Cannot Be Disrupted by 200-Fold Molar Excess of TW-37.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1606-1606
Author(s):  
Anton-Scott Goustin ◽  
Anshu Giri ◽  
Shaomeng Wang ◽  
Ayad Al-Katib ◽  
Mohammad M. Ramzi
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Small Molecule ◽  
Protein Interaction ◽  
Plasmon Resonance ◽  
Dissociation Constants ◽  
Drug Binding ◽  
Mitochondrial Outer Membrane ◽  
Multidomain Proteins ◽  
Hydrophobic Groove

Abstract Control of the intrinsic apoptotic pathway is exercised through the interplay of a 20-member family of proteins related to the founding member, Bcl-2. These proteins are confined to the vicinity of two membrane surfaces, the endoplasmic reticulum and the mitochondrial outer membrane. Like most of cellular biochemistry, reactions are confined in nano time and space which do not allow the approach to equilibrium seen at the macro level of the test tube, motivating us to initiate a dynamic systems biology approach to these non-equilibrium reactions. Our laboratory has investigated the mechanism of action of a non-peptide small molecule TW-37, which represents a new chemical class of compounds like ABT-737 (Abbott) useful in the induction of apoptosis in tumor cells. The drug induces apoptotic death in lymphoma cells with an observed EC50 of 290 nM (Mohammad et al., Clin. Cancer Res.13, 2226 (2007)). TW-37 binds with nanomolar affinity to the hydrophobic groove on the surface of the pro-survival Bcl-2 proteins which can accommodate the hydrophobic face of the amphipathic α-helix of BH3-only family members such as Bid. To model how the drug perturbs the Bcl-2 system, we have immobilized a highly-helical 24-mer Bid-derived BH3-domain peptide via its N-terminus to the surface of a CM5 chip (ligand) and analyzed peptide-protein interaction with flowing analyte proteins Bcl-2, Bcl-XL, Mcl-1, Bcl-w, and Bfl-1/A1 using the Biacore 3000. Using an analysis of Langmuir binding, we observe that the analyte proteins bind Bid with ON-rates (ka) varying modestly (3.01–7.72 E+05 1/Ms), but dissociate from Bid with 17-fold variation in OFF-rate (kd). The Bcl-w complex with Bid is the slowest to dissociate with a kd of 0.0015 (1/s); the most rapid dissociation is seen with Bcl-2 and Bid, where the kd is 0.0385. We find that Bid interacts with the multidomain proteins with dissociation constants (KD) between 4 and 82 nM, intermediate between earlier determinations (J. Med. Chem.49, 6139 (2006)); Molec. Cell17, 393 (2005)), but significantly lower than the KD for the drug TW-37 and the multidomain proteins (260–1100 nM reported previously; 79-333 nM measured here). Contrary to the suggestion of Shangary and Johnson (Biochemistry41, 9485 (2002)), our BH3-mimetic TW-37 is unable to disrupt already-formed Bid heterodimers with the mutidomain survival proteins; when heterodimers on the Biacore chip are challenged with doses of TW-37 up to 10000 nM. Consequently, the only opportunity the drug might have to perturb the Bcl-2 system is to locate the hydrophobic groove before Bid. Thus, a new picture emerges of drug action in which the dynamics of pre-apoptotic protein-protein interactions creates a temporal window for drug binding to the hydrophobic groove. In this light, attempts to develop small molecule inhibitors of protein-protein interaction (Current Topics in Medicinal Chem7(10), 922–927 (2007)) should incorporate surface plasmon resonance measurements of ka and kd into preclinical drug discovery programs in addition to KD estimates of drug binding to target. Figure Figure

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