Surface plasmon resonance: a useful technique for cell biologists to characterize biomolecular interactions

Surface plasmon resonance (SPR) is a powerful technique for monitoring the affinity and selectivity of biomolecular interactions. SPR allows for analysis of association and dissociation rate constants and modeling of biomolecular interaction kinetics, as well as for equilibrium binding analysis and ligand specificity studies. SPR has received much use and improved precision in classifying protein–protein interactions, as well as in studying small-molecule ligand binding to receptors; however, lipid–protein interactions have been underserved in this regard. With the field of lipids perhaps the next frontier in cellular research, SPR is a highly advantageous technique for cell biologists, as newly identified proteins that associate with cellular membranes can be screened rapidly and robustly for lipid specificity and membrane affinity. This technical perspective discusses the conditions needed to achieve success with lipid–protein interactions and highlights the unique lipid–protein interaction mechanisms that have been elucidated using SPR. It is intended to provide the reader a framework for quantitative and confident conclusions from SPR analysis of lipid–protein interactions.     

Role of non-electrostatic forces in antimicrobial potency of a dengue-virus derived fusion peptide VG16KRKP: Mechanistic insight into the interfacial peptide-lipid interactions

Cationic antimicrobial peptides (AMPs) are emerging as effective alternatives to conventional therapeutics that are used against the ever-rising number of multidrug-resistant microbial strains. Most studies established the peptide's amphipathicity and electrostatic interaction with the membrane as the basis for their antimicrobial effect. However, the interplay between the stoichiometric ratio of lipids, local geometry, diverse physicochemical properties of the host membranes and antimicrobial peptide efficacy is still poorly understood. In the present study, we investigate the mechanism of interaction of VG16KRKP (VARGWKRKCPLFGKGG), a novel AMP designed from the dengue-virus fusion peptide, with bacterial/fungal membrane mimics. Fluorescence based dye leakage assays show that membrane disruption is not solely induced by electrostatic interaction but also driven by stoichiometric ratio of the lipids that dictates the net surface charge, amount of lipid defects and local geometry of the membrane. Solid-state ¹⁴N and ³¹P NMR experiments show that peptide interaction results in lowering of lipid order around both the headgroups and acyl chains, suggesting deep peptide insertion. Further, an increase or decrease in membrane stability of the host membrane was observed in differential scanning calorimetry (DSC) thermograms, dictated by the overall stoichiometric ratio of the lipids and the sterol present. In general, our results help understand the diverse fates of host membranes against an antimicrobial peptide.     

Surface Plasmon Resonance Imaging Sensors: A Review

Surface plasmon resonance (SPR) imaging sensors realize label-free, real-time, highly sensitive, quantitative, high-throughput biological interaction monitoring and the binding profiles from multi-analytes further provide the binding kinetic parameters between different biomolecules. In the past two decades, SPR imaging sensors found rapid increasing applications in fundamental biological studies, medical diagnostics, drug discovery, food safety, precision measurement, and environmental monitoring. In this paper, we review the recent advances of SPR imaging sensor technology towards high-throughput multi-analyte screening. Finally, we describe our multiplex spectral-phase SPR imaging biosensor for high-throughput biosensing applications.     

Real-time monitoring of odorant-induced cellular reactions using surface plasmon resonance

Surface plasmon resonance (SPR) is a powerful technique for measuring molecular interaction in real-time. SPR can be used to detect molecule to cell interactions as well as molecule to molecule interactions. In this study, the SPR-based biosensing technique was applied to real-time monitoring of odorant-induced cellular reactions. An olfactory receptor, OR I7, was fused with a rho-tag import sequence at the N-terminus of OR I7, and expressed on the surface of human embryonic kidney (HEK)-293 cells. These cells were then immobilized on a SPR sensor chip. The intensity of the SPR response was linearly dependent on the amount of injected odorant. Among all the aldehyde containing odorants tested, the SPR response was specifically high for octanal, which is the known cognate odorant for the OR I7. This SPR response is believed to have resulted from intracellular signaling triggered by the binding of odorant molecules to the olfactory receptors expressed on the cell surface. This SPR system combined with olfactory receptor-expressed cells provides a new olfactory biosensor system for selective and quantitative detection of volatile compounds.     

Transient Potential Gradients and Impedance Measures of Tethered Bilayer?Lipid Membranes: Pore-Forming Peptide Insertion and the Effect of?Electroporation

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.     

α-Synuclein Senses Lipid Packing Defects and Induces Lateral Expansion of Lipids Leading to Membrane Remodeling

There is increasing evidence for the involvement of lipid membranes in both the functional and pathological properties of α-synuclein (α-Syn). Despite many investigations to characterize the binding of α-Syn to membranes, there is still a lack of understanding of the binding mode linking the properties of lipid membranes to α-Syn insertion into these dynamic structures. Using a combination of an optical biosensing technique and in situ atomic force microscopy, we show that the binding strength of α-Syn is related to the specificity of the lipid environment (the lipid chemistry and steric properties within a bilayer structure) and to the ability of the membranes to accommodate and remodel upon the interaction of α-Syn with lipid membranes. We show that this interaction results in the insertion of α-Syn into the region of the headgroups, inducing a lateral expansion of lipid molecules that can progress to further bilayer remodeling, such as membrane thinning and expansion of lipids out of the membrane plane. We provide new insights into the affinity of α-Syn for lipid packing defects found in vesicles of high curvature and in planar membranes with cone-shaped lipids and suggest a comprehensive model of the interaction between α-Syn and lipid bilayers. The ability of α-Syn to sense lipid packing defects and to remodel membrane structure supports its proposed role in vesicle trafficking.     

New trends in instrumental design for surface plasmon resonance-based biosensors

Surface plasmon resonance (SPR)-based biosensing is one of the most advanced label free, real time detection technologies. Numerous research groups with divergent scientific backgrounds have investigated the application of SPR biosensors and studied the fundamental aspects of surface plasmon polaritons that led to new, related instrumentation. As a result, this field continues to be at the forefront of evolving sensing technology. This review emphasizes the new developments in the field of SPR-related instrumentation including optical platforms, chips design, nanoscale approach and new materials. The current tendencies in SPR-based biosensing are identified and the future direction of SPR biosensor technology is broadly discussed.     

Aptamer–Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay

The goal of this work is to explore the amplification effect of aptamer–gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer–Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer–Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer–Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer–Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer–Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.     

Binding properties of antimicrobial agents to dipeptide terminal of lipid II using surface plasmon resonance

We developed a surface plasmon resonance (SPR) assay to estimate the interactions of antimicrobial agents with the dipeptide terminal of lipid II (d-alanyl-d-alanine) and its analogous dipeptides (l-alanyl-l-alanine and d-alanyl-d-lactate) as ligands. The established SPR method showed the reproducible immobilization of ligands on sensor chip and analysis of binding kinetics of antimicrobial agents to ligands. The ligand-immobilized chip could be used repeatedly for at least 200 times for the binding assay of antimicrobial agents, indicating that the ligand-immobilized chip is sufficiently robust for the analysis of binding kinetics. In this SPR system, the selective and specific binding characteristics of vancomycin and its analogs to the ligands were estimated and the kinetic parameters were calculated. The kinetic parameters revealed that one of the remarkable binding characteristics was the specific interaction of vancomycin to only the d-alanyl-d-alanine ligand. In addition, the kinetic binding data of SPR showed close correlation with the antimicrobial activity. The SPR data of other antimicrobial agents (e.g., teicoplanin) to the ligands showed correlation with the antimicrobial activity on the basis of the therapeutic mechanism. Our SPR method could be a valuable tool for predicting the binding characteristics of antimicrobial agents to the dipeptide terminal of lipid II.     

Peptide‐based therapeutic cancer vaccine: Current trends in clinical application

The peptide‐based therapeutic cancer vaccines have attracted enormous attention in recent years as one of the effective treatments of tumour immunotherapy. Most of peptide‐based vaccines are based on epitope peptides stimulating CD8⁺ T cells or CD4⁺ T helper cells to target tumour‐associated antigens (TAAs) or tumour‐specific antigens (TSAs). Some adjuvants and nanomaterials have been exploited to optimize the efficiency of immune response of the epitope peptide to improve its clinical application. At present, numerous peptide‐based therapeutic cancer vaccines have been developed and achieved significant clinical benefits. Similarly, the combination of peptide‐based vaccines and other therapies has demonstrated a superior efficacy in improving anti‐cancer activity. We delve deeper into the choices of targets, design and screening of epitope peptides, clinical efficacy and adverse events of peptide‐based vaccines, and strategies combination of peptide‐based therapeutic cancer vaccines and other therapies. The review will provide a detailed overview and basis for future clinical application of peptide‐based therapeutic cancer vaccines.     

The membrane-binding properties of a class A amphipathic peptide

The membrane-binding properties of a class A amphipathic peptide (18D) were investigated using two different immobilized model membrane systems. The first system involved the use of surface plasmon resonance (SPR) to study the binding of 18D to dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylglycerol (DMPG), which allowed peptide binding to be monitored in real time. The SPR experiments indicated stronger binding of 18D to DMPG than DMPC, which kinetic analysis revealed was due to a faster on-rate. The second model membrane system involved immobilized membrane chromatography in which the binding of 18D to either DMPC or DMPG monolayers covalently linked to silica particles was analysed by elution chromatography. Stronger binding affinity of 18D was also obtained with the negatively charged phosphatidylglycerol (PG) monolayer compared to the phosphatidylcholine (PC) monolayer, which was consistent with the SPR results. Non-linear binding behaviour of 18D to the immobilized lipid monolayers was also observed, which suggests that the peptide undergoes conformational and orientational changes upon binding to the immobilized PC and PG ligands. Significant band broadening was also observed on both monolayers, with larger bandwidths obtained on the PC surface, indicating slower binding and orientation kinetics with the zwitterionic surface. The dependence of logk' on the percentage of methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while at lower temperatures, electrostatic and other polar forces also made a contribution to the affinity of the peptides for the lipid monolayer particularly. Overall, these results demonstrate the complementary use of these two lipid biosensors which allows the role of hydrophobic and electrostatic forces in peptide–membrane interactions to be studied and insight gained into the kinetic factors associated with these interactions.     

ProPose: a docking engine based on a fully configurable protein–ligand interaction model

Virtual high-throughput screening of molecular databases and in particular high-throughput protein–ligand docking are both common methodologies that identify and enrich hits in the early stages of the drug design process. Current protein–ligand docking algorithms often implement a program-specific model for protein–ligand interaction geometries. However, in order to create a platform for arbitrary queries in molecular databases, a new program is desirable that allows more manual control of the modeling of molecular interactions.For that reason, ProPose, an advanced incremental construction docking engine, is presented here that implements a fast and fully configurable molecular interaction and scoring model. This program uses user-defined, discrete, pharmacophore-like representations of molecular interactions that are transformed on-the-fly into a continuous potential energy surface, allowing for the incorporation of target specific interaction mechanisms into docking protocols in a straightforward manner. A torsion angle library, based on semi-empirical quantum chemistry calculations, is used to provide minimum energy torsion angles for the incremental construction algorithm. Docking results of a diverse set of protein–ligand complexes from the Protein Data Bank demonstrate the feasibility of this new approach.As a result, the seamless integration of pharmacophore-like interaction types into the docking and scoring scheme implemented in ProPose opens new opportunities for efficient, receptor-specific screening protocols. Figure ProPose — a fully configurable protein-ligand docking program — transforms pharmacophores into a smooth potential energy surface.This revised version was published online in October 2004 with corrections to the Graphical Abstract.     

Differential scanning calorimetry and X-ray diffraction studies of the specificity of the interaction of antimicrobial peptides with membrane-mimetic systems

Interest in biophysical studies on the interaction of antimicrobial peptides and lipids has strongly increased because of the rapid emergence of antibiotic-resistant bacterial strains. An understanding of the molecular mechanism(s) of membrane perturbation by these peptides will allow a design of novel peptide antibiotics as an alternative to conventional antibiotics. Differential scanning calorimetry and X-ray diffraction studies have yielded a wealth of quantitative information on the effects of antimicrobial peptides on membrane structure as well as on peptide location. These studies clearly demonstrated that antimicrobial peptides show preferential interaction with specific phospholipid classes. Furthermore, they revealed that in addition to charge-charge interactions, membrane curvature strain and hydrophobic mismatch between peptides and lipids are important parameters in determining the mechanism of membrane perturbation. Hence, depending on the molecular properties of both lipid and peptide, creation of bilayer defects such as phase separation or membrane thinning, pore formation, promotion of nonlamellar lipid structures or bilayer disruption by the carpet model or detergent-like action, may occur. Moreover, these studies suggest that these different processes may represent gradual steps of membrane perturbation. A better understanding of the mutual dependence of these parameters will help to elucidate the molecular mechanism of membrane damage by antimicrobial peptides and their target membrane specificity, keys for the rationale design of novel types of peptide antibiotics.     

Integrating Solid-State NMR and Computational Modeling to Investigate the Structure and Dynamics of Membrane-Associated Ghrelin

The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide’s secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide’s positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8–17 form an α-helix, while residues 21–23 and 26–27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane.     

The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics

The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.     

Transient Potential Gradients and Impedance Measures of Tethered Bilayer Lipid Membranes: Pore-Forming Peptide Insertion and the Effect of Electroporation

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.     

Tertiary motifs as building blocks for the design of protein‐binding peptides

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.     

Smart design of fiber optic surfaces for improved plasmonic biosensing

Although the phenomenon of surface plasmon resonance (SPR) is known for more than a century now, traditional prism-based SPR platforms have hardly escaped the research laboratories despite being recognized for the sensitive and specific performance. Significant efforts have been made over the last years to overcome their existing limitations by coupling the SPR phenomenon to the fiber optic (FO) technology. While this platform has been promoted as cost-effective and simpler alternative capable of handling label-free bioassays, quantification and real-time monitoring of biomolecular interactions, examples of its applicability in sensing and biosensing remain to date very limited. The FO-SPR system is still in development and requires further advancements for reaching the stability and sensitivity of the benchmark SPR systems. Among existing strategies for device improvement, those based on modifying the FO tips using nanomaterials are mostly studied. These small-scale objects provide a wide range of possibilities for alternating the architecture of the FO sensitive zone, enabling also unique effects such as localized SPR (LSPR). This mini-review summarizes the latest innovations in the fabrication procedures which use nanoparticles or other nanomaterials, aiming at FO-SPR technology performance improvements, as well as addition of new device features and functionalities.     

Sensing peptide–oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein

We present a new methodology for site-specific sensing of peptide–oligonucleotide (ODN) interactions using a solvatochromic fluorescent label based on 3-hydroxychromone (3HC). This label was covalently attached to the N-terminus of a peptide corresponding to the zinc finger domain of the HIV-1 nucleocapsid protein (NC). On interaction with target ODNs, the labeled peptide shows strong changes in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from the bulk water by the ODN bases. Remarkably, this two-color response depends on the ODN sequence and correlates with the 3D structure of the corresponding complexes, suggesting that the 3HC label monitors the peptide–ODN interactions site-specifically. By measuring the two-color ratio, we were also able to determine the peptide–ODN-binding parameters and distinguish multiple binding sites in ODNs, which is rather difficult using other fluorescence methods. Moreover, this method was found to be more sensitive than the commonly used steady-state fluorescence anisotropy, especially in the case of small ODNs. The described methodology could become a new universal tool for investigating peptide–ODN interactions.