Mechanisms underlying drug-mediated regulation of membrane protein function

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. Drug regulation of membrane protein function thus may be mediated by both direct interactions with the protein and drug-induced alterations of bilayer properties, in which the latter will alter the energetics of protein conformational changes. To tease apart these mechanisms, we examine how the prototypical, proton-gated bacterial potassium channel KcsA is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). All tested drugs inhibited KcsA activity, and the changes in the different gating steps varied with bilayer thickness, suggesting a coupling to the bilayer. Examining the correlations between changes in KcsA gating steps and bilayer properties reveals that drug-induced regulation of membrane protein function indeed involves bilayer-mediated mechanisms. Both direct, either specific or nonspecific, binding and bilayer-mediated mechanisms therefore are likely to be important whenever there is overlap between the concentration ranges at which a drug alters membrane protein function and bilayer properties. Because changes in bilayer properties will impact many diverse membrane proteins, they may cause indiscriminate changes in protein function.

Isolating Specific vs. Non-Specific Binding Responses in Conducting Polymer Biosensors for Bio-Fingerprinting

A longstanding challenge for accurate sensing of biomolecules such as proteins concerns specifically detecting a target analyte in a complex sample (e.g., food) without suffering from nonspecific binding or interactions from the target itself or other analytes present in the sample. Every sensor suffers from this fundamental drawback, which limits its sensitivity, specificity, and longevity. Existing efforts to improve signal-to-noise ratio involve introducing additional steps to reduce nonspecific binding, which increases the cost of the sensor. Conducting polymer-based chemiresistive biosensors can be mechanically flexible, are inexpensive, label-free, and capable of detecting specific biomolecules in complex samples without purification steps, making them very versatile. In this paper, a poly (3,4-ethylenedioxyphene) (PEDOT) and poly (3-thiopheneethanol) (3TE) interpenetrating network on polypropylene–cellulose fabric is used as a platform for a chemiresistive biosensor, and the specific and nonspecific binding events are studied using the Biotin/Avidin and Gliadin/G12-specific complementary binding pairs. We observed that specific binding between these pairs results in a negative ΔR with the addition of the analyte and this response increases with increasing analyte concentration. Nonspecific binding was found to have the opposite response, a positive ΔR upon the addition of analyte was seen in nonspecific binding cases. We further demonstrate the ability of the sensor to detect a targeted protein in a dual-protein analyte solution. The machine-learning classifier, random forest, predicted the presence of Biotin with 75% accuracy in dual-analyte solutions. This capability of distinguishing between specific and nonspecific binding can be a step towards solving the problem of false positives or false negatives to which all biosensors are susceptible.

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection

Aims: We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on prostate-specific antigen (PSA). Background: High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based enzyme-linked lectin sorbent assays (ELLSAs), as they can mask specific binding signals and cause false-positive results. Methods: In this study, we constructed an ELLSA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELLSA. Results: The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELLSA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. Conclusion: The ELLSA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.

Unexpected Enhancement of Antimicrobial Polymer Activity against Staphylococcus aureus in the Presence of Fetal Bovine Serum

Cationic and amphiphilic polymers are known to exert broad-spectrum antibacterial activity by a putative mechanism of membrane disruption. Typically, nonspecific binding to hydrophobic components of the complex biological milieu, such as globular proteins, is considered a deterrent to the successful application of such polymers. To evaluate the extent to which serum deactivates antibacterial polymethacrylates, we compared their minimum inhibitory concentrations in the presence and absence of fetal bovine serum. Surprisingly, we discovered that the addition of fetal bovine serum (FBS) to the assay media in fact enhances the antimicrobial activity of polymers against Gram-positive bacteria S. aureus, whereas the opposite is the case for Gram-negative E. coli. Here, we present these unexpected trends and develop a hypothesis to potentially explain this unusual phenomenon.

Novel UV-activated biofunctionalization of up-converting nanocrystals for detection of proteins

Lanthanide doped nanocrystals capable to emit higher energy photons under excitation with lower energy radiation are promising for a broad range of applications including biodetection, biosensing, and bioimaging. However, the adaptation of these nanoparticles to the biological environment that requires good water-solubility, stability and ease of further functionalization still remains a challenge. The application of nanoparticles for biodetection or in various assays encountered many difficulties arising mainly from the strong tendency of nanoparticles for aggregation or nonspecific binding. Here we present a new method to obtain soluble and stable in water-based buffers NaYF4:Yb3+Er3+ nanocrystals with modified surface ready for further conjugation with biomolecules. In the presented approach polyvinylpyrrolidone/vinyl alcohol copolymer (PVP/VA) with photo-activatable linker (N-5-Azido-2-nitrobenzoyl group—ANB-NOS) was used for initial coating due to its high non-covalent affinity to nanoparticles surface. Subsequent coating with aminated dextran by ultraviolet light activation of ANB-NOS was carried out. This step has a significant impact on nanocrystals stability in the physiological buffer as well as on protein conjugation. Conjugation of biomolecules was possible by employing another photo-linker—sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA). Bacterial Protein G has been selected to create a universal immune-imaging agent due to its ability to bind Fc fragment of most mammalian immunoglobulins. Moreover, the direct attachment of antibodies to nanoparticles was also examined. The activity of biofunctionalized nanocrystals was tested in immunoblot test, that confirmed preserved activity of attached molecules and lack of undesired nonspecific binding or precipitation on the assay membrane. Graphic abstract

Response to Comment on “Tumor-initiating cells establish an IL-33–TGF-β niche signaling loop to promote cancer progression”

Kamphuis et al. argue that macrophages accumulated in the proximity of tumor-initiating cells do not express the high-affinity immunoglobulin E receptor FcεRIα. Although we cannot exclude the possibility of nonspecific binding of anti-FcεRIα antibody (clone MAR-1), we provide evidence that macrophages in squamous cell carcinomas express FcεRIα and that IL-33 induces FcεRIα expression in bone marrow cell–derived macrophages.

A Bit Stickier, a Bit Slower, a Lot Stiffer: Specific vs. Nonspecific Binding of Gal4 to DNA

Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding—equilibrium and kinetics—by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4–DNA interactions. We found a strong enthalpy–entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.