Affinity Measurement of Non-covalent Interactions of the Covalent KRAS G12C GDP Inhibitor MRTX849 to RAS Isoforms Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is an optical effect at an electron-rich surface that enables affinity measurements of biomolecules in real time. It is label free and versatile, not limited to proteins, nucleic acids, and small molecules. SPR is a widely accepted method to measure not only affinity of molecular interactions but also association and dissociation rates of such interactions. In this chapter, we describe a general method to measure the affinity of a small molecule drug, MRTX849, to GDP bound HRAS, KRAS, and NRAS.

Optical fiber immunosensors based on surface plasmon resonance for the detection of Escherichia coli.

Every year, millions of people suffer some form of illness associated with the consumption of contaminated food. Escherichia coli (E. coli), found in the intestines of humans and other animals, is commonly associated with various diseases, due to the existence of pathogenic strains. Strict monitoring of food products for human consumption is essential to ensure public health, but traditional cell culture-based methods are associated with long waiting times and high costs. New approaches must be developed to achieve cheap, fast, and on-site monitoring. Thus, in this work, we developed optical fiber sensors based on surface plasmon resonance. Gold and cysteamine-coated fibers were functionalized with anti-E. coli antibody and tested using E. coli suspensions with concentrations ranging from 1 cell/mL to 105 cells/mL. An average logarithmic sensitivity of 0.21 ± 0.01 nm/log(cells/mL) was obtained for three independent assays. An additional assay revealed that including molybdenum disulfide resulted in an increase of approximately 50% in sensitivity. Specificity and selectivity were also evaluated, and the sensors were used to analyze contaminated water samples, which verified their promising applicability in the aquaculture field.

Numerical analysis and early detection of Plasmodium falciparum using a high performance plasmonic biosensor with an external sensing approach.

The authors propose a surface plasmon resonance (SPR) sensor based on photonic crystal fibers (PCFs) using three hexagonal ring lattices. The sensor can detect biomolecules with maximum wavelength and amplitude sensitivities of 23,000 nm/RIU and 1310.93R I U -1, respectively, in the RI range of 1.32 to 1.42. It can detect infected red blood cells with Plasmodium falciparum for RIs of 1.402, 1.373, 1.395, and 1.383 in various malaria-infected red blood cell stages, including ring phase, trophozoite phase, and schizont phase. Furthermore, the sensor will be able to detect biomolecules such as viruses, proteins, DNA/RNA strands, acetone, ethanol, hexane, isopropanol, hexanol, formic acid, allyl cyanide, and others in its range. With these impressive results and identification capacity, the proposed sensor would benefit the biomaterial field and be appropriate for the early identification of malaria disease.

Efficient Sequential Detection of Two Antibiotics Using a Fiber-Optic Surface Plasmon Resonance Sensor.

Antibiotic residues have become a worldwide public safety issue. It is vital to detect multiple antibiotics simultaneously using sensors. A new and efficient method is proposed for the combined detection of two antibiotics (enrofloxacin (Enro) and ciprofloxacin (Cip)) in milk using surface plasmon resonance (SPR) sensors. Based on the principle of immunosuppression, two antibiotic antigens (for Enro and Cip) were immobilized on an optical fiber surface with conjugates of bovine serum albumin using dopamine (DA) polymerization. Each single antigen was bound to its corresponding antibody to derive standard curves for Enro and Cip. The fiber-optic sensor's sensitivity was 2900 nm/RIU. Detection limits were calculated to be 1.20 ng/mL for Enro and 0.81 ng/mL for Cip. The actual system's recovery rate was obtained by testing Enro and Cip in milk samples; enrofloxacin's and ciprofloxacin's mean recoveries from the milk samples were 96.46-120.46% and 96.74-126.9%, respectively. In addition, several different regeneration solutions were tested to analyze the two target analytes' regeneration ability; NaOH and Gly-HCl solutions were found to have the best regeneration ability.

Using the Photo-Piezoelectric Effect of AuPt@BaTiO3 Oxidase Mimetics for Colorimetric Detection of GSH in Serum.

Nanozymes possess major advantages in catalysis and biosensing compared with natural nanozymes. In this study, the AuPt@BaTiO3 bimetallic alloy Schottky junction is prepared to act as oxidase mimetics, and its photo-piezoelectric effect is investigated. The synergy between the photo-piezoelectric effect and the local surface plasmon resonance enhances the directional migration and separation of photogenerated electrons, as well as hot electrons induced by the AuPt bimetallic alloy. This synergy significantly improves the oxidase-like activity. A GSH colorimetric detection platform is developed based on this fading principle. Leveraging the photo-piezoelectric effect allows for highly sensitive detection with a low detection limit (0.225 µM) and reduces the detection time from 10 min to 3 min. The high recovery rate (ranging from 99.91% to 101.8%) in actual serum detection suggests promising potential for practical applications. The development of bimetallic alloy heterojunctions presents new opportunities for creating efficient nanozymes.

SPR and Double Resonance LPG Biosensors for Helicobacter pylori BabA Antigen Detection.

Given the medical and social significance of Helicobacter pylori infection, timely and reliable diagnosis of the disease is required. The traditional invasive and non-invasive conventional diagnostic techniques have several limitations. Recently, opportunities for new diagnostic methods have appeared based on the recent advance in the study of H. pylori outer membrane proteins and their identified receptors. In the present study we assess the way in which outer membrane protein-cell receptor reactions are applicable in establishing a reliable diagnosis. Herein, as well as in other previous studies of ours, we explore the reliability of the binding reaction between the best characterized H. pylori adhesin BabA and its receptor, the blood antigen Leb. For the purpose we developed surface plasmon resonance (SPR) and double resonance long period grating (DR LPG) biosensors based on the BabA-Leb binding reaction for diagnosing H. pylori infection. In SPR detection, the sensitivity was estimated at 3000 CFU/mL-a much higher sensitivity than that of the RUT test. The DR LPG biosensor proved to be superior in terms of accuracy and sensitivity-concentrations as low as 102 CFU/mL were detected.

Antibody-independent surface plasmon resonance assays for influenza vaccine quality control.

Surface plasmon resonance (SPR)-based biosensors have emerged as a powerful platform for bioprocess monitoring due to their ability to detect biointeractions in real time, without the need for labeling. Paramount for the development of a robust detection platform is the immobilization of a ligand with high specificity and affinity for the in-solution species of interest. Following the 2009 H1N1 pandemic, much effort has been made toward the development of quality control platforms for influenza A vaccine productions, many of which have employed SPR for detection. Due to the rapid antigenic drift of influenza's principal surface protein, hemagglutinin, antibodies used for immunoassays need to be produced seasonally. The production of these antibodies represents a 6-8-week delay in immunoassay and, thus, vaccine availability. This review focuses on SPR-based assays that do not rely on anti-HA antibodies for the detection, characterization, and quantification of influenza A in bioproductions and biological samples. KEY POINTS: • The single radial immunodiffusion assay (SRID) has been the gold standard for the quantification of influenza vaccines since 1979. Due to antigenic drift of influenza's hemagglutinin protein, new antibody reagents for the SRID assay must be produced each year, requiring 6-8 weeks. The resulting delay in immunoassay availability is a major bottleneck in the influenza vaccine pipeline. This review highlights ligand options for the detection and quantification of influenza viruses using surface plasmon resonance biosensors.

Plasmonic Fluorescence Sensors in Diagnosis of Infectious Diseases.

The increasing demand for rapid, cost-effective, and reliable diagnostic tools in personalized and point-of-care medicine is driving scientists to enhance existing technology platforms and develop new methods for detecting and measuring clinically significant biomarkers. Humanity is confronted with growing risks from emerging and recurring infectious diseases, including the influenza virus, dengue virus (DENV), human immunodeficiency virus (HIV), Ebola virus, tuberculosis, cholera, and, most notably, SARS coronavirus-2 (SARS-CoV-2; COVID-19), among others. Timely diagnosis of infections and effective disease control have always been of paramount importance. Plasmonic-based biosensing holds the potential to address the threat posed by infectious diseases by enabling prompt disease monitoring. In recent years, numerous plasmonic platforms have risen to the challenge of offering on-site strategies to complement traditional diagnostic methods like polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). Disease detection can be accomplished through the utilization of diverse plasmonic phenomena, such as propagating surface plasmon resonance (SPR), localized SPR (LSPR), surface-enhanced Raman scattering (SERS), surface-enhanced fluorescence (SEF), surface-enhanced infrared absorption spectroscopy, and plasmonic fluorescence sensors. This review focuses on diagnostic methods employing plasmonic fluorescence sensors, highlighting their pivotal role in swift disease detection with remarkable sensitivity. It underscores the necessity for continued research to expand the scope and capabilities of plasmonic fluorescence sensors in the field of diagnostics.

Network pharmacology, computational biology integrated surface plasmon resonance technology reveals the mechanism of ellagic acid against rotavirus.

The target and mechanism of ellagic acid (EA) against rotavirus (RV) were investigated by network pharmacology, computational biology, and surface plasmon resonance verification. The target of EA was obtained from 11 databases such as HIT and TCMSP, and RV-related targets were obtained from the Gene Cards database. The relevant targets were imported into the Venny platform to draw a Venn diagram, and their intersections were visualized. The protein-protein interaction networks (PPI) were constructed using STRING, DAVID database, and Cytoscape software, and key targets were screened. The target was enriched by Gene Ontology (GO) and KEGG pathway, and the 'EA anti-RV target-pathway network' was constructed. Schrodinger Maestro 13.5 software was used for molecular docking to determine the binding free energy and binding mode of ellagic acid and target protein. The Desmond program was used for molecular dynamics simulation. Saturation mutagenesis analysis was performed using Schrodinger's Maestro 13.5 software. Finally, the affinity between ellagic acid and TLR4 protein was investigated by surface plasmon resonance (SPR) experiments. The results of network pharmacological analysis showed that there were 35 intersection proteins, among which Interleukin-1ß (IL-1ß), Albumin (ALB), Nuclear factor kappa-B1 (NF-κB1), Toll-Like Receptor 4 (TLR4), Tumor necrosis factor alpha (TNF-α), Tumor protein p53 (TP53), Recombinant SMAD family member 3 (SAMD3), Epidermal growth factor (EGF) and Interleukin-4 (IL-4) were potential core targets of EA anti-RV. The GO analysis consists of biological processes (BP), cellular components (CC), and molecular functions (MF). The KEGG pathways with the highest gene count were mainly related to enteritis, cancer, IL-17 signaling pathway, and MAPK signaling pathway. Based on the crystal structure of key targets, the complex structure models of TP53-EA, TLR4-EA, TNF-EA, IL-1ß-EA, ALB-EA, NF-κB1-EA, SAMD3-EA, EGF-EA, and IL-4-EA were constructed by molecular docking (XP mode of flexible docking). The MMGBS analysis and molecular dynamics simulation were also studied. The Δaffinity of TP53 was highest in 220 (CYS → TRP), 220 (CYS → TYR), and 220 (CYS → PHE), respectively. The Δaffinity of TLR4 was highest in 136 (THR → TYR), 136 (THR → PHE), and 136 (THR → TRP). The Δaffinity of TNF-α was highest in 150 (VAL → TRP), 18 (ALA → GLU), and 144 (PHE → GLY). SPR results showed that ellagic acid could bind TLR4 protein specifically. TP53, TLR4, and TNF-α are potential targets for EA to exert anti-RV effects, which may ultimately provide theoretical basis and clues for EA to be used as anti-RV drugs by regulating TLR4/NF-κB related pathways.

Surface plasmon resonance-enhanced photoelectrochemical immunoassay with Cu-doped porous Bi2WO6 nanosheets.

The development of rapid screening sensing platforms to improve pre-screening mechanisms in community healthcare is necessary to meet the significant need for portable testing in biomarker diagnostics. Here, we designed a portable smartphone-based photoelectrochemical (PEC) immunoassay for carcinoembryonic antigen (CEA) detection using Cu-doped ultrathin porous Bi2WO6 (CuBWO) nanosheets as the photoactive material. The CuBWO nanosheets exhibit a fast photocurrent response and excellent electrical transmission rate under UV light due to their surface plasmon resonance effect (SPR). The method uses glucose oxidase-labeled secondary antibody as a signal indicator for sandwich-type immune conjugation. In the presence of the target CEA, the electrons and holes generated at the surface of the photo-excited ultrathin porous CuBWO were rapidly consumed by the production of H2O2 from glucose oxidase oxidizing glucose, resulting in a weakened photocurrent signal. The photocurrent intensity increased logarithmically and linearly with increasing CEA concentration (0.02-50 ng mL-1), with a detection limit of 15.0 pg mL-1 (S/N = 3). The system provides a broader idea for inferring the electron-hole transport mechanism in ultrathin porous nanosheet layer materials and developing efficient PEC sensors.

Precision Loading and Delivery of Molecular Cargo by Size-Controlled Coacervation of Gold Nanoparticles Functionalized with Elastin-like Peptides.

Thermoresponsive elastin-like peptides (ELPs) have been extensively investigated in biotechnology and medicine, but little attention has been paid to the process by which coacervation causes ELP-decorated particles to aggregate. Using gold nanoparticles (AuNPs) functionalized with a cysteine-terminated 96-repeat of the VPGVG sequence (V96-Cys), we show that the size of the clusters that reversibly form above the ELP transition temperature can be finely controlled in the 250 to 930 nm range by specifying the concentration of free V96-Cys in solution and using AuNPs of different sizes. We further find that the localized surface plasmon resonance peak of the embedded AuNPs progressively red-shifts with cluster size, likely due to an increase in particle-particle contacts. We exploit this fine control over size to homogeneously load precise amounts of the dye Nile Red and the antibiotic Tetracycline into clusters of different hydrodynamic diameters and deliver cargos near-quantitatively by deconstructing the aggregates below the ELP transition temperature. Beyond establishing a key role for free ELPs in the agglomeration of ELP-functionalized particles, our results provide a path for the thermally controlled delivery of precise quantities of molecular cargo. This capability might prove useful in combination photothermal therapies and theranostic applications, and to trigger spatially and temporally uniform responses from biological, electronic, or optical systems.

A sensitivity-enhanced plasmonic sensing platform modified with Co(OH)2 nanosheets.

In the detection of biomolecules, surface plasmon resonance (SPR) sensors require high sensitivity. In this study, we propose a sensitivity-enhanced functionalized plasmonic interface based on Ag-TiO2-Co(OH)2 nanosheets structure. Compared to unmodified SPR sensors, the sensitivity of the sensor decorated with TiO2 and Co(OH)2 nanosheets is increased by 130.84%, reaching 5764.27 nm/RIU. This enhancement is attributed to the high refractive index of the coating, as well as the high specific surface area and abundant active sites provided by the synthesized Co(OH)2 nanosheets with a multi-grooved structure. Additionally, employing a double-antibody sandwich method, the antibody-functionalized plasmonic interface enables specific detection of human serum albumin (HSA). The linear response of this sensor was in the wide range of 0.4-150 µM, and the LOD reached 154.89 nM(KD is approximately 1.73 × 10-6 M). This novel SPR sensor offers a new strategy for biochemical sensing and provides a highly sensitive platform for immunoassays.

Regulation of band gap and localized surface plasmon resonance by loading Au nanorods on violet phosphene nanosheets for photodynamic/photothermal synergistic anti-infective therapy.

In the face of the serious threat to human health and the economic burden caused by bacterial antibiotic resistance, 2D phosphorus nanomaterials have been widely used as antibacterial agents. Violet phosphorus nanosheets (VPNSs) are an exciting bandgap-adjustable 2D nanomaterial due to their good physicochemical properties, yet the study of VPNS-based antibiotics is still in its infancy. Here, a composite of gold nanorods (AuNRs) loaded onto VPNS platforms (VPNS/AuNR) is constructed to maximize the potential of VPNSs for antimicrobial applications. The loading with AuNRs not only enhances the photothermal performance via a localized surface plasmon resonance (LSPR) effect, but also enhances the light absorption capacity due to the narrowing of the band gap of the VPNSs, thus increasing the ROS generation capacity. The results demonstrate that VPNS/AuNR exhibits outstanding antibacterial properties and good biocompatibility. Attractively, VPNS/AuNR is then extensively tested for treating skin wound infections, suggesting promising in vivo antibacterial and wound-healing features. Our findings may open a novel direction to develop a versatile VPNS-based treatment platform, which can significantly boost the progress of VPNS exploration.

Protein-Protein Binding Kinetics by Biolayer Interferometry.

The specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, immobilization, the amount of sample required, the number of measurements that can be accomplished simultaneously, or the cost. Bio-Layer Interferometry (BLI) is a label-free biophysical method to measure the kinetics of protein-protein interactions. Label-free interaction assays are a broad family of methods that do not require protein modifications (other than immobilization) or labels such as fusions with fluorescent proteins or transactivating domains or chemical modifications like biotinylation or reaction with radionuclides. Besides BLI, other label-free techniques that are widely used for determining protein-protein interactions include surface plasmon resonance (SPR), thermophoresis, and isothermal titration calorimetry (ITC), among others.

Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design.

The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

Detection of insulin oligomeric forms by a novel surface plasmon resonance-diffusion coefficient based approach.

Insulin is commonly used to treat diabetes and undergoes aggregation at the site of repeated injections in diabetic patients. Moreover, aggregation is also observed during its industrial production and transport and should be avoided to preserve its bioavailability to correctly adjust glucose levels in diabetic patients. However, monitoring the effect of various parameters (pH, protein concentration, metal ions, etc.) on the insulin aggregation and oligomerization state is very challenging. In this work, we have applied a novel Surface Plasmon Resonance (SPR)-based experimental approach to insulin solutions at various experimental conditions, monitoring how its diffusion coefficient is affected by pH and the presence of metal ions (copper and zinc) with unprecedented sensitivity, precision, and reproducibility. The reported SPR method, hereby applied to a protein for the first time, besides giving insight into the insulin oligomerization and aggregation phenomena, proved to be very robust for determining the diffusion coefficient of any biomolecule. A theoretical background is given together with the software description, specially designed to fit the experimental data. This new way of applying SPR represents an innovation in the bio-sensing field and expanding the potentiality of commonly used SPR instruments well over the canonical investigation of biomolecular interactions.

Programming Intracellular Clustering of Spiky Nanoparticles via Liposome Encapsulation.

The intracellular clustering of anisotropic nanoparticles is crucial to the improvement of the localized surface plasmon resonance (LSPR) for phototherapy applications. Herein, we programmed the intracellular clustering process of spiky nanoparticles (SNPs) by encapsulating them into an anionic liposome via a frame-guided self-assembly approach. The liposome-encapsulated SNPs (lipo-SNPs) exhibited distinct and enhanced lysosome-triggered aggregation behavior while maintaining excellent monodispersity, even in acidic or protein-rich environments. We explored the enhancement of the photothermal therapy performance for SNPs as a proof of concept. The photothermal conversion efficiency of lipo-SNPs clusters significantly increased 15 times compared to that of single lipo-SNPs. Upon accumulation in lysosomes with a 2.4-fold increase in clustering, lipo-SNPs resulted in an increase in cell-killing efficiency to 45% from 12% at 24 µg/mL. These findings indicated that liposome encapsulation provides a promising approach to programing nanoparticle clustering at the target site, which facilitates advances in the development of smart nanomedicine with programmable enhancement in LSPR.

Study of cell and drug interactions based on dual-mode detection using SPR and fluorescence imaging.

The investigation of the interactions between cells and drugs forms a crucial aspect of biological and clinical medical studies. Generally, single-cell or local-cellular studies require a microscopic imaging system with high magnifications, which suffers from low detection throughputs and poor time responses. The study presented in this paper combined SPR and fluorescence to achieve cell localization, real-time monitoring of cell images and quantitative analysis of drugs. In order to obtain more comprehensive, accurate and real-time data, a dual-mode system based on surface plasmon resonance (SPR) and fluorescence was constructed based on a 4× magnification lens. This enables simultaneous studies of an entire cell and a specific region of the cell membrane. An adaptive adjustment algorithm was established for distorted SPR images, achieving temporal and spatial matching of the dual-mode detection. The combination of SPR and fluorescence not only achieved micro-detection but also complemented the qualitative or quantitative limitations of SPR or fluorescence method alone. In system characterization, the response signal of SPR was noticed to increase with the increasing concentration of EGF in stimulated cells. It indicated that this platform could be employed for quantitative detection of the cell membrane region. Upon addition of EGF, a peak in the SPR curve was observed, and the cells in the corresponding SPR image turned whiter. This indicated that the platform can simultaneously monitor the SPR response signal and image changes. The response time of fluorescence in EGF testing was several seconds earlier than SPR, revealing that signal transduction first occurred in the whole cell and then propagated to the cell membrane region. The inhibitory ability of Gefitinib on cells was verified in a fast and real-time manner within 20 min. The results indicated that the detection limit of this method was 20 IU/mL for EGF and 10 µg/mL for Gefitinib. In conclusion, this study demonstrates the advantages of SPR and fluorescence dual-mode techniques in the analysis of cell-drug interactions, as well as their strong potential in drug screening.

A two-step mechanism for the binding of the HIV-1 MPER epitope by the 10E8 antibody onto biosensor-supported lipid bilayers.

HIV-1 antibodies targeting the carboxy-terminal area of the membrane-proximal external region (ctMPER) are close to exerting viral pan-neutralization. Here, we reconstituted the ctMPER epitope as the N-terminal extremity of the Env glycoprotein transmembrane domain helix and immobilized it onto biosensor-supported lipid bilayers. We assessed the binding mechanism of anti-MPER antibody 10E8 through Surface Plasmon Resonance, and found, through equilibrium and kinetic binding analyses as a function of bilayer thickness, peptide length, and paratope mutations, that 10E8 engages first with the epitope peptide (encounter), limited by ctMPER helix accessibility at the membrane surface, and then inserts into the lipid bilayer assisted by favorable Fab-membrane interactions (docking). This mechanistic information may help in devising new strategies to develop more efficient MPER-targeting vaccines.

The Multienzyme Complex Nature of Dehydroepiandrosterone Sulfate Biosynthesis.

Dehydroepiandrosterone (DHEA), a precursor of steroid sex hormones, is synthesized by steroid 17-alpha-hydroxylase/17,20-lyase (CYP17A1) with the participation of microsomal cytochrome b5 (CYB5A) and cytochrome P450 reductase (CPR), followed by sulfation by two cytosolic sulfotransferases, SULT1E1 and SULT2A1, for storage and transport to tissues in which its synthesis is not available. The involvement of CYP17A1 and SULTs in these successive reactions led us to consider the possible interaction of SULTs with DHEA-producing CYP17A1 and its redox partners. Text mining analysis, protein-protein network analysis, and gene co-expression analysis were performed to determine the relationships between SULTs and microsomal CYP isoforms. For the first time, using surface plasmon resonance, we detected interactions between CYP17A1 and SULT2A1 or SULT1E1. SULTs also interacted with CYB5A and CPR. The interaction parameters of SULT2A1/CYP17A1 and SULT2A1/CYB5A complexes seemed to be modulated by 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Affinity purification, combined with mass spectrometry (AP-MS), allowed us to identify a spectrum of SULT1E1 potential protein partners, including CYB5A. We showed that the enzymatic activity of SULTs increased in the presence of only CYP17A1 or CYP17A1 and CYB5A mixture. The structures of CYP17A1/SULT1E1 and CYB5A/SULT1E1 complexes were predicted. Our data provide novel fundamental information about the organization of microsomal CYP-dependent macromolecular complexes.