Interaction kinetics of peptide lipids-mediated gene delivery.

BACKGROUND: During the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency. RESULTS: As cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp (- 0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in HeLa cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplex was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the "proton sponge effect" of the lipid. CONCLUSIONS: The interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.

Mutation-induced protein interaction kinetics changes affect apoptotic network dynamic properties and facilitate oncogenesis.

It has been a consensus in cancer research that cancer is a disease caused primarily by genomic alterations, especially somatic mutations. However, the mechanism of mutation-induced oncogenesis is not fully understood. Here, we used the mitochondrial apoptotic pathway as a case study and performed a systematic analysis of integrating pathway dynamics with protein interaction kinetics to quantitatively investigate the causal molecular mechanism of mutation-induced oncogenesis. A mathematical model of the regulatory network was constructed to establish the functional role of dynamic bifurcation in the apoptotic process. The oncogenic mutation enrichment of each of the protein functional domains involved was found strongly correlated with the parameter sensitivity of the bifurcation point. We further dissected the causal mechanism underlying this correlation by evaluating the mutational influence on protein interaction kinetics using molecular dynamics simulation. We analyzed 29 matched mutant-wild-type and 16 matched SNP--wild-type protein systems. We found that the binding kinetics changes reflected by the changes of free energy changes induced by protein interaction mutations, which induce variations in the sensitive parameters of the bifurcation point, were a major cause of apoptosis pathway dysfunction, and mutations involved in sensitive interaction domains show high oncogenic potential. Our analysis provided a molecular basis for connecting protein mutations, protein interaction kinetics, network dynamics properties, and physiological function of a regulatory network. These insights provide a framework for coupling mutation genotype to tumorigenesis phenotype and help elucidate the logic of cancer initiation.

Tractable RNA-ligand interaction kinetics.

BACKGROUND: The binding of small ligands to RNA elements can cause substantial changes in the RNA structure. This constitutes an important, fast-acting mechanism of ligand-controlled transcriptional and translational gene regulation implemented by a wide variety of riboswitches. The associated refolding processes often cannot be explained by thermodynamic effects alone. Instead, they are governed by the kinetics of RNA folding. While the computational analysis of RNA folding can make use of well-established models of the thermodynamics of RNA structures formation, RNA-RNA interaction, and RNA-ligand interaction, kinetic effects pose fundamentally more challenging problems due to the enormous size of the conformation space. The analysis of the combined process of ligand binding and structure formation even for small RNAs is plagued by intractably large state spaces. Moreover, the interaction is concentration-dependent and thus is intrinsically non-linear. This precludes the direct transfer of the strategies previously used for the analysis of RNA folding kinetics. RESULTS: In our novel, computationally tractable approach to RNA-ligand kinetics, we overcome the two main difficulties by applying a gradient-based coarse graining to RNA-ligand systems and solving the process in a pseudo-first order approximation. The latter is well-justified for the most common case of ligand excess in RNA-ligand systems. We present the approach rigorously and discuss the parametrization of the model based on empirical data. The method supports the kinetic study of RNA-ligand systems, in particular at different ligand concentrations. As an example, we apply our approach to analyze the concentration dependence of the ligand response of the rationally designed, artificial theophylline riboswitch RS3. CONCLUSION: This work demonstrates the tractability of the computational analysis of RNA-ligand interaction. Naturally, the model will profit as more accurate measurements of folding and binding parameters become available. Due to this work, computational analysis is available to support tasks like the design of riboswitches; our analysis of RS3 suggests strong co-transcriptional effects for this riboswitch. The method used in this study is available online, cf. Section "Availability of data and materials".

Kinetic analysis of the weak affinity interaction between tris and lysozyme.

The biosensor based on total internal reflection imaging ellipsometry (TIRIE), regarded as an automotive real-time research approach for biomolecular interaction, is introduced to analyze the kinetic process of the weak interaction between tris and lysozyme. The experiment is performed by delivering lysozyme solution diluted to different concentrations to the biosensor substrate interface immobilized with tris. By applying pseudo-first-order interaction kinetics model, we are able to obtain the kinetic parameters from fitting experimental data. The calculated association rate constant and dissociation rate constant of tris and lysozyme interaction are in 10(-2) mol(-1) s(-1) and 10(3)s(-1) magnitude, respectively. To further improve TIRIE's ability for kinetically characterizing biomolecular interaction, a theoretical method to deduce associate rate constant before experiment is proposed.

Effect of molecular crowders on ligand binding kinetics with G-quadruplex DNA probed by fluorescence correlation spectroscopy.

Guanine-rich single-stranded DNA folds into G-quadruplex DNA (GqDNA) structures, which play crucial roles in various biological processes. These structures are also promising targets for ligands, potentially inducing antitumor effects. While thermodynamic parameters of ligand/DNA interactions are well-studied, the kinetics of ligand interaction with GqDNA, particularly in cell-like crowded environments, remain less explored. In this study, we investigate the impact of molecular crowding agents (glucose, sucrose, and ficoll 70) at physiologically relevant concentrations (20% w/v) on the association and dissociation rates of the benzophenoxazine-core based ligand, cresyl violet (CV), with human telomeric antiparallel-GqDNA. We utilized fluorescence correlation spectroscopy (FCS) along with other techniques. Our findings reveal that crowding agents decrease the binding affinity of CV to GqDNA, with the most significant effect-a nearly three-fold decrease-observed with ficoll 70. FCS measurements indicate that this decrease is primarily due to a viscosity-induced slowdown of ligand association in the crowded environment. Interestingly, dissociation rates remain largely unaffected by smaller crowders, with only small effect observed in presence of ficoll 70 due to direct but weak interaction between the ligand and ficoll. These results along with previously reported data provide valuable insights into ligand/GqDNA interactions in cellular contexts, suggesting a conserved mechanism of saccharide crowder influence, regardless of variations in GqDNA structure and ligand binding mode. This underscores the importance of considering crowding effects in the design and development of GqDNA-targeted drugs for potential cancer treatment.

Recent Advances in the Quantitative Determination of Protein Receptor-Ligand Interaction Kinetics.

Receptor-ligand binding, which is crucial to a variety of biomedical and biochemical processes, immune responses, and signal transduction, forms the basis for many biotechnological applications. The specific binding between a receptor and a ligand is the beginning of the biological function of the receptor or ligand molecule. Therefore, a summary study of methods for quantitative determination of receptor-ligand interaction kinetics is necessary. In this review, the kinetic parameters that traditionally describe the pattern of receptor-ligand interactions are first introduced. We then summarize and analyze methods for quantitative determination of receptor-ligand interaction kinetics, including direct kinetic measurements, cytology-based measurements, computational chemistry-based measurements, and single-molecule force spectrometry technique measurements of receptor-ligand interactions. Direct measurements of the kinetics of receptor-ligand interactions are further described in methods based on surface plasmon resonance, surface-enhanced Raman spectroscopy, photoelectrochemistry, mass spectrometry binding analyses, nuclear magnetic resonance technology, and electrochemical methods. This review provides a comprehensive and accurate description of the kinetic studies of protein receptor-ligand interactions.

Electrochemical biosensor for the evaluation of monoclonal antibodies targeting the N protein of SARS-CoV-2 virus.

The emergence of COVID-19 caused by the coronavirus SARS-CoV-2 has prompted a global pandemic that requires continuous research and monitoring. This study presents a design of an electrochemical biosensing platform suitable for the evaluation of monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein. Screen-printed carbon electrodes (SPCE) modified with gold nanostructures (AuNS) were applied to design a versatile and sensitive sensing platform. Electrochemical techniques, including electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), were used to investigate the interactions between immobilised recombinant N (rN) protein and several monoclonal antibodies (mAbs). The electrochemical characterisation of SPCE/AuNS/rN demonstrated a successful immobilisation of rN, enhancing the electron transfer kinetics. Affinity interactions between immobilised rN and four mAbs (mAb-4B3, mAb-4G6, mAb-12B2, and mAb-1G5) were explored. Although mAb-4B3 showed some non-linearity, the other monoclonal antibodies exhibited specific and well-defined interactions followed by the formation of an immune complex. The biosensing platform demonstrated high sensitivity in the linear range (LR) from 0.2 nM to 1 nM with limits of detection (LOD) ranging from 0.012 nM to 0.016 nM for mAb-4G6, mAb-12B2, and mAb-1G5 and limits of quantification (LOQ) values ranging from 0.035 nM to 0.139 nM, as determined by both EIS and SWV methods. These results highlight the system's potential for precise and selective detection of monoclonal antibodies specific to the rN. This electrochemical biosensing platform provides a promising route for the sensitive and accurate detection of monoclonal antibodies specific to the rN protein.

Antibody Profiling: Kinetics with Native Biomarkers for Diagnostic Assay and Drug Developments.

Despite remarkable progress in applied Surface Plasmon Resonance (SPR)-based methods, concise monitoring of kinetic properties for native biomarkers from patient samples is still lacking. Not only are low concentrations of native targets in patient samples, often in the pM range, a limiting and challenging factor, but body fluids as complex matrices furthermore complicate measurements. The here-described method enables the determination of kinetic constants and resulting affinities for native antigens from patients' cerebrospinal fluid (CSF) and sera binding to antibodies. Using a significantly extended target-enrichment step, we modified a common sandwich-assay protocol, based on a primary and secondary antibody. We successfully analyze antibody kinetics of native targets from a variety of origins, with consistent results, independent of their source. Moreover, native neurofilament light chain (NFL) was investigated as an exemplary biomarker. Obtained data reveal antibodies recognizing recombinant NFL with high affinities, while showing no, or only significantly weakened binding to native NFL. The indicated differences for recombinant vs. native material demonstrate another beneficial application. Our assay is highly suitable for gaining valuable insights into characteristics of native biomarkers, thus impacting on the binder development of diagnostic reagents or pharmaceutical drugs.

Protein Interaction Analysis by Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is an optical technique that is utilized for detecting molecular interactions that occur in direct protein-protein interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected, after polarized light impinges upon the surface, is altered and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. One of the advantages in SPR is its high sensitivity, compatible with the need for purification of small amounts of protein for analysis. This chapter concentrates on practical methodologies for performing surface plasmon resonance analysis.

Accounting for Interaction Kinetics between Gold Nanoparticles and Aptamers Enables High-Performance Colorimetric Sensors.

DNA aptamers have emerged as promising probes for challenging analytes that cannot be easily detected by conventional probes, including small-molecule targets. Among the different signal transduction approaches, gold nanoparticle (AuNP) aggregation assays have been widely used to generate a colorimetric response from aptamer-target interactions. This sensor design relies on the competition between the aptamer adsorbing to the AuNP surface versus interacting with the target, whereby target binding reduces the number of adsorbed aptamers that destabilizes AuNPs toward salt-induced aggregation, thereby inducing a color change. However, this thermodynamic framework overlooks the potential influence of interaction kinetics of different aptamer conformations with AuNP surfaces and with targets in solution or near surfaces. Here, we show that aptamers become more strongly adsorbed on AuNPs over time, and these trapped aptamers are less responsive toward the target analyte. By varying the sequence of addition in sensing assays, we demonstrate that these interaction kinetics have a significant effect on the sensor response and thereby produce an effective sensor for methamphetamine (meth) at biologically relevant levels in oral fluids. Along with underpinning new tools for assay development, this new knowledge also highlights the need for aptamer selection strategies that evolve aptamer sequences based on the functionality that they need to exhibit in an actual sensor.

Detection of Exosomes Using Total Internal Reflected Imaging Ellipsometry.

Exosomes are a kind of membrane-bound phospholipid nanovesicle that are secreted extensively in a variety of biological fluids. Accumulating evidence has indicated that exosomes not only communicate with cells, but also perform functional roles in physiology and pathology. In addition, exosomes have also elicited a great deal of excitement due to their potential as disease biomarkers. Therefore, requirements for sensitive methods capable of precisely and specifically determining exosomes were needed. Herein, we not only develop a sensing surface to capture exosomes but also compare two surface proteins on exosomes, which are appropriate for detecting exosome surface markers by total internal reflected imaging ellipsometry (TIRIE). Protein G and antibody were immobilized on a thin layer of golden substrate to form the biosensing surface. The bio-interaction between antibodies and exosomes was recorded by the TIRIE in real time. The distance between exosomes adhered on a surface was 44 nm ± 0.5 nm. The KD  of anti-CD9 and exosome was lower than anti-CD63 and exosome by introducing pseudo-first-order interaction kinetics, which suggested that CD9 is more suitable for exosome surface markers than CD63. The limit of detection (LOD) of TIRIE was 0.4 µg/mL. In conclusion, we have proposed a surface for the detection of exosomes based on TIRIE, which can make the detection of exosomes convenient and efficient.

Interaction kinetics and accessibility of sulfadiazine in model clay-humic acid suspension: Electron spin resonance investigations with nitroxide spin label.

The characterization of the interaction of sulfonamides with soil is of particular interest in environmental risk and persistence assessment. In the present work electron spin resonance spectroscopy (ESR) was used to investigate the interaction kinetics of spin labelled sulfadiazine (SL-SDZ) with model clay-humic acid suspensions. The ESR spectra showed that SL-SDZ incubated with Leonardite humic acid (LHA) and Ca-hectorite as model clay was immobilized due to covalent binding of its aniline moiety to LHA. From the immobilization kinetics measured over a period of 1200 h a pseudo-first order reaction with a time constant of 82.6 ± 25.0 h of covalent binding was determined. Additionally, SL-SDZ was strongly sorbed by LHA immediately after incubation but not durably sequestered. Compared to incubation without Ca-hectorite the covalent binding kinetics of SL-SDZ as well as its strong sorption were retarded.

Evaluation of affinity sensor response kinetics towards dimeric ligands linked with spacers of different rigidity: Immobilized recombinant granulocyte colony-stimulating factor based synthetic receptor binding with genetically engineered dimeric analyte derivatives.

The modelling of protein-protein binding kinetics is important for the development of affinity-sensors and the prediction of signaling protein based drug efficiency. Therefore, in this research we have evaluated the binding kinetics of several genetically designed protein models: (i) three different ligands based on granulocyte colony-stimulating factor GCSF homo-dimeric derivatives linked by differed by linkers of different length and flexibility; (ii) an antibody-like receptor (GCSF-R) based on two GCSF-receptor sites immobilized to Fc domains, which are common parts of protein structures forming antibodies. Genetically engineered GCSF-R is similar to an antibody because it, like the antibody, has two binding sites, which both selectively bind with GCSF ligands. To design the affinity sensor model studied here, GCSF-R was immobilized on a thin gold layer via self-assembled monolayer conjugated with Protein-G. Binding kinetics between immobilized GCSF-R and all three different recombinant GCSF-based homo-dimeric derivatives were evaluated by total internal reflection ellipsometry. Association constants were determined by fitting mathematical models to the experimental data. It was clearly observed that both (i) affinity and (ii) binding kinetics depend on the length and flexibility of the linker that connects both domains of a GCSF-based ligand. The fastest association between immobilized GCSF-R and GCSF-based ligands was observed for ligands whose GCSF domains were interconnected by the longest and the most flexible linker. Here we present ellipsometry-based measurements and models of the interaction kinetics that advance the understanding of bidentate-receptor-based immunosensor action and enables us to predict the optimal linker structure for the design of GCSF-based medications.

A bombykol electrochemical receptor sensor and its kinetics.

This study aimed to explore the interaction between bombykol and BmOR1 and also provide a paradigm for agroforestry pest control. The electrochemical biosensor signal amplification system was used: nanogold with horseradish peroxidase. An electrochemical bilayer nanogold membrane receptor sensor was developed using the following schemes and processes: twice self-assembly of nanogold and succeeding absorption of Bombyx mori olfactory receptor 1 (BmOR1); sex pheromone-binding protein; spectral scanning and transmission electron microscope to characterize nanogold sol; and atomic force microscope, cyclic voltammetry, and AC impedance methods to characterize individual processes of sensor assembly. The amperometric I-T curve was adopted to measure the response current upon interaction with different concentrations of bombykol (diluted in phosphate-buffered saline) and BmOR1. The results demonstrated the receptor-ligand interaction pattern, which was similar to enzymatic reaction kinetics, with the activation constant Ka of up to 8.57 × 10-20 mol/L and signal magnification of about 10,000-fold. In this study, the simulation of intracellular receptor signaling cascade by an electrochemical signal amplification system helped in directly measuring BmOR1-bombykol ligand interaction and exploring the kinetics after the self-assembly of BmOR1 on the biosensor. It provided a novel platform for future studies on receptor-ligand interaction.

Analytical, thermodynamical and kinetic characteristics of photoluminescence immunosensor for the determination of Ochratoxin A.

Ochratoxin A (OTA) is one of the most widespread and dangerous food contaminants. Therefore, rapid, label-free and precise detection of low OTA concentrations requires novel sensing elements with advanced bio-analytical properties. In the present paper we report photoluminescence (PL) based immunosensor for the detection of OTA. During the development of immunosensor photoluminescent ZnO nanorods (ZnO-NRs) were deposited on glass substrate. Then the ZnO-NRs were silanized and covalently modified by Protein-A (Glass/ZnO-NRs/Protein-A). The latest structure was modified by antibodies against OTA (Anti-OTA) in order to form OTA-selective layer (Glass/ZnO-NRs/Protein-A/Anti-OTA). In order to improve immunosensors selectivity the surface of Glass/ZnO-NRs/Protein-A/Anti-OTA was additionally blocked by BSA. Formed Glass/ZnO-NRs/Protein-A/BSA&Anti-OTA structures were integrated within portable fiber optic detection system, what is important for the development of low cost and portable immunosensors. The immunosensor has been tested in a wide range of OTA concentrations from 10-4ng/ml until 20ng/ml. Interaction isotherms were derived from analytical signals of immunosensor. Association constant and Gibbs free energy for the interaction of Glass/ZnO-NRs/Protein-A/Anti-OTA with OTA were calculated, analyzed and compared with some other related results. Sensitivity range and limit of detection were determined as 0.1-1ng/ml and 10-2ng/ml, respectively. Interaction kinetics of ZnO-NRs with OTA was evaluated. Response time of the immunosensor toward OTA was in the range of 500-800s. Some insights related to the mechanism of PL-signal generation are proposed and discussed.

Binding and structure-kinetic relationship analysis of selective TLR4-targeted immunosuppressive self-assembling heparin nanoparticles.

Self-assembling aliphatic heparin derivatives were shown to inhibit the immune system by antagonizing Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD2). In the present study, glycol split heparin-d-erythro-sphingosine conjugates (NAHNP) and its regioselectively desulfated derivatives with shortened aliphatic chains were investigated regarding their biophysical properties in the interaction with TLR4/MD2. Two-dimensional nuclear Overhauser effect spectroscopy studies showed that upon glycol splitting, the heparin backbone gains extra adaptability that facilitates binding to proteins. However, unlike native heparin or glycol split non-anticoagulant heparin (NAH), hydrophobic derivatization of NAH forces sulfated iduronic acid residues to change configuration from a 2S0 skew-boat to a 1C4 chair form. Whereas neither heparin nor NAH had any appreciable effect, NAHNP significantly inhibited lipopolysaccharide-induced activation of the NF-κB transcription factor. We showed that NAHNP binds to TLR4/MD2 with an affinity of 62.3 nM. In line with computational studies, biosensor-based structure-kinetic relationship studies demonstrated that 6-O-sulfo groups of d-glucosamine residue were essential in binding to arginines of both TLR4 and MD2 domains of the receptor complex. The desulfation of 6-O-sulfo groups decreases the association kinetics from 4.2 × 104 M-1 s-1 to 3.8 × 103 M-1 s-1, which results in a decreased affinity of 800 nM. Two aliphatic chains of NAHNP bound to the MD2 pocket similarly to lipopolysaccharide. A decrease in chain length resulted in a loss of inhibitory activity on NF-κB transcription and binding affinity to TLR4/MD2. In conclusion, the present study characterizes the immunosuppressive effect of aliphatic heparin derivatives and provides a promising strategy to develop selective immunosuppressants for acute and chronic inflammatory disorders.

Nanofluidic Fluorescence Microscopy (NFM) for real-time monitoring of protein binding kinetics and affinity studies.

Kinetic monitoring of protein interactions offers insights to their corresponding functions in cellular processes. Surface plasmon resonance (SPR) is the current standard tool used for label-free kinetic assays; however, costly and sophisticated setups are required, decreasing its accessibility to research laboratories. We present a cost-effective nanofluidic-based immunosensor for low-noise real-time kinetic measurement of fluorescent-labeled protein binding. With the combination of fluorescence microscopy and reversed buffer flow operation, association and dissociation kinetics can be accessed in one single experiment without extra buffer loading step, which results in a simplified operation and reduced time of analysis compared to typical microfluidic immunoassays. Kinetic constants of two representative protein-ligand binding pairs (streptavidin/biotin; IgG/anti-IgG) were quantified. The good agreement of extracted rate constants with literature values and analogous SPR measurements indicates that this approach is applicable to study protein interactions of medium- and high-affinities with a limit of detection down to 1 pM, regardless of the analyte size.

Thermodynamic analyses of amino acid residues at the interface of an antibody B2212A and its antigen roundabout homolog 1.

Artificial affinity maturation of antibodies is promising but often shows difficulties because the roles of each amino acid residue are not well known. To elucidate their roles in affinity against the antigen and thermal stability, interface residues in single-chain Fv of an antibody B2212A with its antigen roundabout homolog 1 were mutated and analyzed. Some amino acids played important roles in the affinity while others contributed to thermal stability.

Real-time label-free detection and kinetic analysis of Etanercept-Protein A interactions using quartz crystal microbalance.

A quartz crystal microbalance (QCM) was constructed to assess if such a biosensor has value as a complementary real-time label-free analysis platform for the biopharmaceutical industry. This was achieved through modifying QCM crystals with a low-fouling carboxymethyl-dextran layer bearing Protein A, and then injecting solutions containing Etanercept (i.e., Enbrel®) into the QCM chambers. The kinetics of Enbrel® - Protein A interactions was modeled using the Langmuir binding model and Enbrel® concentrations between 0.75-300ngmL-1. The resulting equilibrium dissociation and association constants (KD and KA) were 5.06×10-8M and 1.98×107M-1, respectively. The association and dissociation rate constants (kon and koff) decreased substantially as Enbrel® concentration, [C], increased, despite that the net binding rate, (kon[C]+koff), increased. The decrease in kon and koff was hypothesized to be a consequence of mass transport limitations. To verify this, QCM dissipation measurements were analyzed to provide insight on solution viscosity. As Enbrel® concentration increased, the net change in dissipation, ΔD, became larger. An augmentation of ΔD is associated with a higher solution viscosity, which would result in an increase in mass transport limitations. Therefore, the decrease in kon and koff for increasing Enbrel® concentration can be attributed to mass transport limitations. In conclusion, QCM is a valuable complementary real-time label-free biosensor analysis platform for the biopharmaceutical industry. Unlike the surface plasmon resonance (SPR) platform, QCM allows measuring dissipation, which can provide insight on how mass transport limitations impact interaction kinetics.

Surface Plasmon Resonance (SPR) Analysis of Binding Interactions of Inner-Ear Proteins.

Surface plasmon resonance is an optical technique that is utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected after polarized light impinges upon the film, is altered, and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. We have utilized surface plasmon resonance to study interaction of proteins of inner-ear sensory epithelia.