Resonance Energy Research Articles

High-fidelity CRISPR/Cas13a trans cleavage-driven assembly of single quantum dot nanosensor for ultrasensitive detection of long noncoding RNAs in clinical breast tissues

Long noncoding RNAs (lncRNAs) act as critical regulators in various cellular processes, and their dysfunction is implicated in carcinogenesis. Herein, we demonstrate high-fidelity CRISPR/Cas13a trans cleavage-driven assembly of single quantum dot (QD) nanosensor for ultrasensitive detection of long noncoding RNAs in clinical tissues. The presence of lncRNA can activate Cas13a/crRNA to collaterally cleave the substrate probes, producing a T7 promoter fragment that can initiate subsequent transcription amplification to generate efficient fluorescence resonance energy transfer (FRET). Taking advantage of excellent specificity of high-fidelity CRISPR/Cas13a system, high efficiency of transcription amplification, and near-zero background of single QD-based FRET, this nanosensor can achieve a detection limit of 1.65 aM, and it can differentiate target lncRNA from its mismatched members with single-base resolution. Moreover, it can measure lncRNA at the single-cell level, distinguish different subtypes of breast cancers, and assess the breast cancer progression. Notably, due to the programmability of crRNAs, this nanosensor can be extended to detect other nucleic acids (e.g., SARS-CoV-2 RNA, circRNA, miRNA, piRNA, and 16S rRNA) by simply altering the spacer region of crRNA, with great potential in lncRNAs-related molecular diagnostics.

Identifying the minimal sets of distance restraints for FRET-assisted protein structural modeling.

Proteins naturally occur in crowded cellular environments and interact with other proteins, nucleic acids, and organelles. Since most previous experimental protein structure determination techniques require that proteins occur in idealized, non-physiological environments, the effects of realistic cellular environments on protein structure are largely unexplored. Recently, Förster resonance energy transfer (FRET) has been shown to be an effective experimental method for investigating protein structure in vivo. Inter-residue distances measured in vivo can be incorporated as restraints in molecular dynamics (MD) simulations to model protein structural dynamics in vivo. Since most FRET studies only obtain inter-residue separations for a small number of amino acid pairs, it is important to determine the minimum number of restraints in the MD simulations that are required to achieve a given root-mean-square deviation (RMSD) from the experimental structural ensemble. Further, what is the optimal method for selecting these inter-residue restraints? Here, we implement several methods for selecting the most important FRET pairs and determine the number of pairs that are needed to induce conformational changes in proteins between two experimentally determined structures. We find that enforcing only a small fraction of restraints, , where is the number of amino acids, can induce the conformational changes. These results establish the efficacy of FRET-assisted MD simulations for atomic scale structural modeling of proteins in vivo.

DNA‐Templated Gold Nanoclusters for miRNA Diagnostics with Branched Hybridization Chain Reaction

In this study, a novel nonlinear branched hybridization chain reaction (BHCR) strategy is developed for accurate biosensing and bioimaging. Four fuel strands are designed. The first two assist the growth of primary long double‐stranded chain. Meanwhile, branched tails are left, which consist the trigger of downstream HCR with the second group of fuel strands. Branched DNA nanostructures are thus formed, which significantly amplify the information of initial target miRNA. Besides, the output fluorescent signal is originated from DNA‐templated gold nanoclusters (AuNCs). Surface ligands of AuNCs always play important roles in the regulation of optical mechanisms. Herein, peptide/DNA‐stabilized AuNCs are prepared. Phosphorothioate‐modified DNA (psDNA) is used as the template for in situ synthesis. The short peptide (Tyr‐Cys‐Tyr, YCY) is introduced as the protecting ligand, which benefits significant Stokes shift via a two‐step Förster resonance energy transfer. Coupling BHCR and AuNCs, this method performs satisfactory for in vitro fluorescent biosensing and intracellular bioimaging. It exhibits potential application for the investigation of miRNA‐related bioprocesses.

Cannabinoid regulation of angiotensin II-induced calcium signaling in striatal neurons.

Calcium ion (Ca2+) homeostasis is crucial for neuron function and neurotransmission. This study focused on the actions mediated by the CB1 receptor (CB1R), the most abundant G protein-coupled receptor (GPCR) in central nervous system (CNS) neurons, over by the AT1R, which is one of the few G protein-coupled CNS receptors able to regulate cytoplasmic Ca2+ levels. A functional interaction suggesting a direct association between these receptors was detected. AT1-CB1 receptor heteromers (AT1CB1Hets) were identified in HEK-293T cells by bioluminescence resonance energy transfer (BRET2). Functional interactions within the AT1-CB1 complex and their potential relevance in Parkinson's disease (PD) were assessed. In situ proximity ligation assays (PLA) identified AT1CB1Hets in neurons, in which an important finding was that Ca2+ level increase upon AT1R activation was reduced in the presence of cannabinoids acting on CB1Rs. AT1CB1Het expression was quantified in samples from the 6-hydroxydopamine (6-OHDA) hemilesioned rat model of PD in which a lower expression of AT1CB1Hets was observed in striatal neurons from lesioned animals (versus non-lesioned). AT1CB1Het expression changed depending on both the lesion and the consequences of levodopa administration, i.e., dyskinesias versus lack of involuntary movements. A partial recovery in AT1CB1Het expression was detected in lesioned animals that developed levodopa-induced dyskinesias. These findings support the existence of a compensatory mechanism mediated by AT1CB1Hets that modulates susceptibility to levodopa-induced dyskinesias in PD. Therefore, cannabinoids may be useful in reducing calcium dyshomeostasis in dyskinesia.

Live-Cell Imaging of miRNAs with the Combination of CHA and HCR Techniques.

The abnormal expression of miRNA-21 is closely related to many cancers, and its sensitive detection plays an important role in the diagnosis and treatment of cancers. In the report, we designed a non-enzymatic amplification sensing system based on the catalytic hairpin assembly (CHA) and palindrome-based hybridization chain reaction (PHCR) technique for the detection and imaging of miRNA in living cells. Based on PHCR technology, two ingeniously designed palindrome-contained fluorescently labeled hairpin probes are sequentially opened upon the stimulation of short oligonucleotide triggers, promoting the autonomous assembly of cross-linked network-like structures (CNSs) and generating fluorescence resonance energy transfer (FRET) signal. Moreover, the PHCR technique can be combined with CHA technology to sufficiently improve amplification efficiency and signal output. By utilizing the CHA-PHCR sensing system, one miRNA-21 activates many triggers through CHA process and in turn initiates the assembly of CNSs by PHCR reaction, efficiently amplifying the FRET signal output. As such, the sensing system can detect target miRNAs down to 10pm with high detection specificity. Moreover, the CNS exhibits the enhanced intracellular stability, enabling the living cell imaging of miRNA-21. The CHA-PHCR sensing system can also screen the expression level of miRNA-21 in different living cells and differentiate cancer cells from healthy cells, which is consistent with the gold-standard PCR method.

Studies of electron-ion resonant recombination of li-like Si11+ions

Abstract Theoretical investigations of L-shell △n = 0, 1, and 2 (2s → 2p, 3 l ′ , and 4 l ′ ) dielectronic recombination (DR), as well as K-shell △n = 1, 2, and 3 (1s → 2 l ′ , 3 l ′ , and 4 l ′ ) DR of Li-like Si 11+ ions are performed, using the relativistic distorted-wave approach. Detailed resonance energies, resonance strengths, and recombination rate coefficients are calculated including complex configuration mixing, Breit interaction, and QED corrections. Good agreement is achieved between the theoretical rate coefficients and the experimental results. The plasma rate coefficients are also calculated for electron temperatures ranging from 10−1 to 106 eV, and an analytical formula is presented to fit the total plasma rate coefficients for convenient modeling of astrophysical and fusion plasmas.

Fluorescent Sensing for the Detection and Quantification of Sulfur-Containing Gases.

Sulfur-containing gases, such as H2S and SO2, play significant roles in a multitude of biological processes affecting human life and health. Precise and efficient detection of these gases is therefore crucial for advancing one's understanding of their biological roles and developing effective diagnostic strategies. Fluorescent sensing offers a highly sensitive and versatile approach for detecting these gases. This Review examines the recent advances in the fluorescent detection of H2S and SO2, highlighting the key mechanisms involved in fluorescence signal transduction, including changes in intensity and wavelength shifts. The diverse array of probe molecules employed for this purpose, including those utilizing mechanisms such as nucleophilic reactions, Förster resonance energy transfer (FRET), and sulfur affinity interactions are explored. In additional to organic sensors, the focus of the Review is particularly directed toward quantum dot (QD) systems, emphasizing their tunable optical properties that hold immense potential for fluorescence sensing. Beyond the traditional III-V QDs, we delve into the emerging fluorescence sensors based on halide perovskite QDs, upconversion nanocrystals, and other novel materials. These advanced QD systems hold promise for the development of highly sensitive and cost-effective gas detectors, paving the way for significant advances in biomedical and environmental monitoring. This Review provides a comprehensive overview of the current state-of-the-art in QD-based fluorescence sensing of sulfur-containing gases and provides a multifaceted discussion comparing organic fluorescent sensors with QD sensors, highlighting the key challenges and opportunities for the integration of fluorescence sensing as it evolves. The Review aims to facilitate further research and development of innovative sensing platforms to enable more accurate and sensitive detection of these important gases.

Probing G-Quadruplexes Conformational Dynamics and Nano-Mechanical Interactions at the Single Molecule Level: Techniques and Perspectives

The analysis of nucleic acid structures, topologies, nano-mechanics and interactions with ligands and other biomacromolecules (most notably proteins) at the single molecule level has become a fundamental topic in molecular biophysics over the last two decades. Techniques such as molecular tweezers, single-molecule fluorescence resonance energy transfer, and atomic force microscopy have enabled us to disclose an unprecedented insight into the mechanisms governing gene replication, transcription and regulation. In this minireview, we survey the main working principles and discuss technical caveats of the above techniques, using as a fil-rouge the history of their achievements in dissecting G-quadruplexes. The revised literature offers a clear example of the superior ability of single-molecule techniques with respect to ensemble techniques to unveil the structural and functional diversity of the several polymorphs corresponding to a single G-quadruplex folding sequence, thus shedding new light on the extreme complexity of these fascinating non-Watson–Crick structures.

An Upconversion Fluorescent Resonant Energy Transfer Biosensor for the Detection of microRNA through DNA Hybridization

An Upconversion Fluorescent Resonant Energy Transfer Biosensor for the Detection of microRNA through DNA Hybridization

Enhanced long-lasting luminescence nanorods for ultrasensitive detection of SARS-CoV-2 N protein

AbstractPersistent luminescence nanomaterials can remain luminescence when the light source is turned off, which exhibits promise in biosensor and bioimaging fields since they have the ability to completely eradicate tissue autofluorescence. Although significant progress has been made in the persistent luminescence biosensing, there is still a dearth of long-afterglow detection platform with low limit of detection (LOD) and high sensitivity. Herein, Zn2GeO4:Mn, Cr persistently luminescent nanorods (PLNRs) with superior persistent luminescence and long afterglow time were developed. The addition of Cr3+ manifestly improves persistent luminescence intensity and afterglow duration through creating a deep defect trap. Then the biosensors were constructed by combining the Zn2GeO4:Mn,Cr PLNRs-antibody and Fe3O4 magnetic nanoparticles (MNPs)-antibody for nucleocapsid protein detection based on electrostatic attraction. The LOD value for nucleocapsid protein realizes as low as 39.82 ag/mL, which is much lower than the previously reported persistent luminescent-based biosensors. Accordingly, the low detection sensitivity is attributed to fluorescence resonance energy transfer. In addition, high specificity is also achieved. Therefore, the as-prepared Zn2GeO4:Mn,Cr persistently luminescent materials can act as the promising candidate in biosensors applications. This strategy provides effective guidance for the development of biosensing platforms with high sensitivity and specificity.

Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein.

Pentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be modified to bind to a specific ssRNA nucleobase. As such, there is interest in developing 'designer' PPRs (dPPRs) for a range of biotechnology applications, including diagnostics or in vivo localization of ssRNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. To address this, we determined the structure of a dPPR bound to its target sequence and used two- and three-color single-molecule fluorescence resonance energy transfer to interrogate the mechanism of ssRNA binding to individual dPPRs in real time. We demonstrate that dPPRs are slower to bind longer ssRNA sequences (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR. Importantly, dPPR binds only to its target sequence (i.e. it does not associate with non-target ssRNA sequences) and does not 'scan' longer ssRNA oligonucleotides for the target sequence. The kinetic constraints imposed by random 3D diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus).

Oriented triplex DNA as a synthetic receptor for transmembrane signal transduction.

Signal transduction across biological membranes enables cells to detect and respond to diverse chemical or physical signals, and replicating these complex biological processes through synthetic methods is of significant interest in synthetic biology. Here we present an artificial signal transduction system using oriented cholesterol-tagged triplex DNA (TD) as synthetic receptors to transmit and amplify signals across lipid bilayer membranes through H+-mediated TD conformational transitions from duplex to triplex. An auxiliary sequence, complementary to the third strand of the TD, ensures a controlled and preferred outward orientation of cholesterol-tagged TD on membranes. Upon external H+ stimuli, the conformational change triggers the translocation of the third strand from the outer to the inner membrane leaflet, resulting in effective transmembrane signal transduction. This mechanism enables fluorescence resonance energy transfer (FRET), selective photocleavage, catalytic signal amplification, and logic gate modulation within vesicles. Our findings demonstrate that these TD-based receptors mimic the functional dynamics of natural G protein-coupled receptors (GPCRs), providing a foundation for advanced applications in biosensing, cell signaling modulation, and targeted drug delivery systems.

FRET verification of crucial interaction sites in RhoA regulation mediated by RhoGDI

AbstractThe small GTPase Rho family are the major factors in mediating actin cytoskeleton dynamics. Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) serve as important negative regulators by complexing with inactive Rho into the cytoplasm. However, how these two molecules interact still needs experimental verification. Based on fluorescence resonance energy transfer (FRET) measurements, we would demonstrate crucial sites in RhoGDI and RhoA for this regulatory role. Cotransfection of RhoGDI markedly reduced RhoA or Cdc42 activity in airway smooth muscle (ASM) cells, while D185R-RhoGDI mutant reversed this decrease, indicating that RhoGDI Asp185 residue is essential for the molecular interaction. R68D-RhoA (mutation in the switch II region) resulted in a deficiency in RhoGDI regulation, while TV37/38NG-RhoA (in the switch I region) displayed low RhoA activity. Hence, the Arg68 site in RhoA is indispensable for regulation by RhoGDI, and Thr37Val38 site is important for maintaining RhoA activity. Additionally, microtubule but not actin cytoskeleton showed inhibitory role in RhoA activity, while the dissolution of either cytoskeleton did not change the regulatory role of RhoGDI. In checking the downstream effect, reduction of RhoA activity induced by PDGF stimulation or RhoGDI decreased cellular stress fibers. In this study, FRET visualization was applied to have experimentally demonstrated the interaction sites and crucial role of RhoGDI in regulating RhoA activity. Graphical Abstract

Activating Ultralong Room-Temperature Phosphorescence of Mono-Ring Arylboronic Acid by Hydrogen Bond for Tunable Afterglow Color and Stimulus Response.

Activating the ultralong room-temperature phosphorescence (RTP) of mono-ring arylboronic acid remains a great challenge, because the capacious free spaces shaped by a rubbery polymeric matrix allow the benzene ring skeleton to freely rotate. Herein, the ultralong RTP in mono-ring arylboronic acid derivatives embedded in a polyvinyl alcohol (PVA) matrix is activated, leveraging enhanced intermolecular and intramolecular hydrogen bonding and activating ultralong RTP. By incorporating diverse PBA derivatives into PVA via click chemistry, 3-aminophenylboronic acid (3A-PBA) doped PVA films showcase the most extended RTP lifetimes (2.24 s) and a high quantum yield (11.2%), alongside rapid and sensitive explosive vapor detection capabilities. These findings not only address the activation challenges of RTP in organic systems by highlighting the crucial role of hydrogen bonding and Förster resonance energy transfer in color tunability, but also mark a significant stride toward overcoming the limitations of current luminescent materials. This work heralds a new era in the development of organic phosphorescent materials, offering a promising strategy for broadening their practical applications and enhancing performance, particularly in environmental monitoring and security.

EXTH-61. TOWARDS A QUANTUM THERAPY FOR GLIOBLASTOMA: A NEW THERAPEUTIC PARADIGM IN MEDICINE

Abstract BACKGROUND A cell operates as an interconnected bioelectrical circuit, utilising electron transfer processes for intracellular communication, with cytochrome c (Cyt c) playing a pivotal role. The redox processes of Cyt c, occurring via electron tunnelling, are essential for its translocation into the cytosol and modulation of its conformation to bind apoptotic protease activating factor 1. This highlights the need for novel technologies capable of interacting with these processes at the atomic scale to control downstream effects and induce apoptosis in cancer cells. METHODS We demonstrate that ‘bio-nanoantennae’, when supplied with an electrical current, enable quantum biological tunnelling for electron transfer (QBET) and facilitate cellular apoptosis in patient-derived IDH wild-type glioblastoma from both the infiltrative tumour margin and proliferative core. The bio-nanoantennae were constructed from gold nanoparticles functionalised with reduced Cyt c and zinc porphyrin as a redox couple. RESULTS Electrical polarisation of these bio-nanoantennae via resonant alternating currents in preclinical glioblastoma cells led to decreased metabolic activity and reduced cell viability by oxidizing Cyt c, thus inducing cellular stress. No significant effect was observed in healthy human astrocyte counterparts. The cytosol localised bio-nanoantennae induced differential gene expression related to ion channels, apoptosis, cancer proliferation and tumour suppression upon activation, in tumour relative to astrocyte cell populations. The bio-nanoantennae were also tested in 3D glioblastoma spheroid models, showing similar effects, and in vivo studies demonstrated a significant reduction in glioblastoma xenograft tumour size. CONCLUSION We propose that bio-nanoantennae modulate the redox state of Cyt c under an electrical field through QBET. To validate this, we investigated the tunnel junction energy and plasmon resonance scattering, and developed a mathematical model to explain the system’s behaviour. This innovative wireless electrical–molecular nanodevice, capable of inducing cancer cell apoptosis, paves the way for further applications of quantum signalling as a new (non-pharmacological) therapeutic paradigm.

BRET Nano Q-Body: A Nanobody-Based Ratiometric Bioluminescent Immunosensor for Point-of-Care Testing.

We developed a nanobody-based homogeneous bioluminescent immunosensor to achieve a one-pot detection for point-of-care testing (POCT). This immunosensor was named BRET nano Q-body as its emission color changes via bioluminescence resonance energy transfer (BRET) upon antigen addition. NanoLuc luciferase and a cysteine-containing tag were fused to the N-terminus of the nanobody, which was labeled with a fluorescent dye via thiol-maleimide Michael addition. The nanobody employed in this proof-of-principle experiment recognizes methotrexate (MTX), a chemotherapeutic agent. After optimizing the fluorescent dye and linker, the BRET nano Q-body dose-dependently exhibited a greater than 7-fold increase in emission ratio (TAMRA/NanoLuc). Moreover, we found its superior thermostability endurance in organic solvents, reducing agents, and detergents due to the robust structure of nanobody, as well as accommodation in biological fluids, such as milk, serum, and whole blood without dilution, with limits of detection of 0.50, 1.6, and 3.7 nM, respectively. Furthermore, the BRET nano Q-body was subjected to lyophilization and fabricated into a paper device, which markedly improved its portability and enabled more than one month of storage at 25 °C. The paper device also performed appropriate functions in the biological fluids without any dilution and can be used for on-site therapeutic drug monitoring of MTX. Altogether, we developed a powerful tool, the BRET nano Q-body, for POCT, and demonstrated its applicability in several biological fluids. In addition, we confirmed the feasibility of paper devices, which are expected to be transformative for in situ detection in therapeutic, diagnostic, and environmental applications.

Early Diagnosis of Tumorigenesis via Ratiometric Carbon Dots with Deep-Red Emissive Fluorescence Based on NAD+ Dependence

The early diagnosis of tumorigenesis is crucial for clinical treatment, but the resolution and sensitivity of conventional short-wavelength biomarkers are not ideal because of the complicated interference in living tissue. Herein, a nicotinamide adenine dinucleotide (NAD+)-responsive probe with deep-red emissive ratiometric fluorescence was synthetized as a promising target for energy metabolism patterns during tumorigenesis. Interestingly, the solvents H3PO4 and 2,2′-dithiodibenzoic acid enhanced the red emission (640 and 680 nm) of o-phenylenediamine-based carbon dots (CDs), leading to the formation of a nanoscale graphite-like skeleton covered with -P=O, -CONH-, -COOH and -NH2 on their surfaces. Meanwhile, this method exhibited high sensitivity to the discriminating target NAD+, with a detection limit of 63 μM due to the inner filter effect and fluorescence resonance energy transfer process between NAD+ and CDs, which is superior to the reported capillary electrophoresis and liquid chromatographic detection methods (the reported detection limit was about 0.2 mM) in complex biological samples and even cancer cells. Encouragingly, NAD+ significantly promoted nucleus-targeting fluorescence and cell migration compared to GSH and pH stimulation, which were gradually eliminated in human hepatocellular carcinoma (HepG2) cells after 2-deoxy-d-Glucose inhibited the glycolytic phenotype. The proposed method holds great potential for the temporal and spatial resolution of NAD+-dependent tumor diagnosis in complex living systems.

Catalytically Active Site Mapping Realized through Energy Transfer Modeling

AbstractThe demands of a sustainable chemical industry are a driving force for the development of heterogeneous catalytic platforms exhibiting facile catalyst recovery, recycling, and resilience to diverse reaction conditions. Homogeneous‐to‐heterogeneous catalyst transitions can be realized through the integration of efficient homogeneous catalysts within porous matrices. Herein, we offer a versatile approach to understanding how guest distribution and evolution impact the catalytic performance of heterogeneous host–guest catalytic platforms by implementing the resonance energy transfer (RET) concept using fluorescent model systems mimicking the steric constraints of targeted catalysts. Using the RET‐based methodology, we mapped condition‐dependent guest (re)distribution within a porous support on the example of modular matrices such as metal–organic frameworks (MOFs). Furthermore, we correlate RET results performed on the model systems with the catalytic performance of two MOF‐encapsulated catalysts used to promote CO2 hydrogenation and ring‐closing metathesis. Guests are incorporated using aperture‐opening encapsulation, and catalyst redistribution is not observed under practical reaction conditions, showcasing a pathway to advance catalyst recyclability in the case of host–guest platforms. These studies represent the first generalizable approach for mapping the guest distribution in heterogeneous host–guest catalytic systems, providing a foundation for predicting and tailoring the performance of catalysts integrated into various porous supports.

Design and implementation of wireless power transfer device

Abstract. Magnetic coupled resonant radio energy transmission (WPT) technology utilizes the coupling of the magnetic fields of the transmitting and receiving coils and wirelessly transmits energy through electromagnetic induction, greatly expanding the applications of power equipment in special environments. The technology uses the magnetic field coupling between the transmitting coil and the receiving coil to realize the wireless transmission of energy through electromagnetic induction. This method breaks the limitation of traditional wire transmission and greatly expands the application potential of power equipment in various special environments. In this paper, an efficient, stable and long-distance wireless charging device is designed and realized. In order to improve transmission efficiency, two-coil configuration with series-series structure is adopted, and its equivalent circuit model is established by circuit theory and Kirchhoff's law. Therefore, a new radio energy transmission device based on single chip microcomputer is designed, including PWM signal generator, level matching circuit, high frequency inverter circuit and other key parts. Expressions for transmission efficiency and output power are derived from equation analysis and power-on experiments performed on a physical device. In addition, the effects of transmission frequency, load impedance and transmission distance on the performance are investigated.

Förster Resonance Energy Transfer Measurements in Living Bacteria for Interaction Studies of BamA with BamD and Inhibitor Identification

The β-barrel assembly machinery (BAM) is a multimeric protein complex responsible for the folding of outer membrane proteins in gram-negative bacteria. It is essential for cell survival and outer membrane integrity. Therefore, it is of impact in the context of antibiotic resistance and can serve as a target for the development of new antibiotics. The interaction between two of its subunits, BamA and BamD, is essential for its function. Here, a FRET-based assay to quantify the affinity between these two proteins in living bacterial cells is presented. The method was applied to identify two interaction hotspots at the binding interface. BamDY184 was identified to significantly contribute to the binding between both proteins through hydrophobic interactions and hydrogen bonding. Additionally, two salt bridges formed between BamDR94, BamDR97, and BamAE127 contributed substantially to the binding of BamA to BamD as well. Two peptides (RFIRLN and VAEYYTER) that mimic the amino acid sequence of BamD around the identified hotspots were shown to inhibit the interaction between BamA and BamD in a dose-dependent manner in the upper micromolar range. These two peptides can potentially act as antibiotic enhancers. This shows that the BamA–BamD interaction site can be addressed for the design of protein–protein interaction inhibitors. Additionally, the method, as presented in this study, can be used for further functional studies on interactions within the BAM complex.