The present invention relates to nanopore sensors and, more specifically, to a design that can be used to detect the specific protein analyte.
Quantitative determination of protein biomarkers is a pressing necessity for disease prognostics, diagnostics, and therapeutics. Recent advancements in quantitative proteomics indicate that there are yet numerous unexplored proteins with potential implications for the progression of pathological conditions. Therefore, there is an increasing need for creating highly specific and sensitive protein sensing elements, which employ rapid signal responses to various biochemical stimuli. Molecular details of protein detection are illuminated using single-molecule technologies. In particular, single-molecule sensing with engineered nanopores using the resistive-pulse technique is adaptable to parallel recording technologies. Despite such a significant benefit, this approach usually requires that the targeted proteins be partitioned into the nanopore interior. Hence, the single-molecule observation is conducted under steric restrictions of the nanopore confinement, impairing the strength of native interactions and, in many cases, limiting its sensing specificity and sensitivity.
Detecting single proteins outside the nanopore is a practical alternative to sample the complexity of protein recognition events without the steric hindrance of the nanopore interior. This task would require a modular fusion protein in which an external protein binder (e.g., receptor) is covalently tethered to a nanopore through a flexible tether. However, a transducing mechanism is required to convert the physical captures and releases of a protein analyte (e.g., its ligand) into a specific electrical signature of the sensor. This challenge is exacerbated by the simple reason that protein binding events must occur away from the pore opening and outside the transmembrane electric field. In addition, changing the system to a new binder-analyte pair necessitates a lengthy and tedious optimization process that includes amplified difficulties. Earlier studies have suggested that these protein sensors may be limited to established protein fragments of ˜100 residues. For example, large protein binders likely induce additional steric constraints, precluding the clearance of the space around the pore opening. Moreover, the interaction interface of the binder must be fully accessible to the protein analyte. Therefore, the heterogeneous architecture, size, charge, and other traits of various protein binders suggest the need for an extensive effort in the protein engineering of an individual sensor for a given protein analyte. Hence, these sensing elements cannot be generalizable to many protein analytes.
The present invention is a new class of sensing elements for probing targeted proteins in solution at a single-detector precision. These sensors have an antibody-mimetic protein scaffold engineered on the tFhuA nanopore, a monomeric β-barrel scaffold. This design preserves the sensor's architecture and high sensitivity and specificity while featuring its generalization to numerous protein analytes. By changing only the binding interface, a novel binder-analyte pair can be obtained and readily implemented into these sensing elements. The binder is a monobody, a recombinant protein based on the 94-residue fibronectin type III domain (the FN3 scaffold). A monobody was fused to the N terminus of tFhuA, on the β-turns side, through a flexible (GGS)2 tether. Using the monobody-based nanopore sensors with varying binding interfaces, it is possible to detect different proteins that vary substantially in charge, size, and structural complexity. When subjected to a challenging heterogeneous solution, this class of nanopore sensors can be used to identify and quantify a protein biomarker in a complex biofluid. Finally, direct measurements of time-resolved protein binding events at adjustable protein concentrations, without the steric hindrance of the nanopore interior, and within a substantially expanded dynamic range will influence nanoproteomics.
In one aspect, the present invention may be a sensor comprises of a lipid membrane having a predetermined membrane potential and a single polypeptide chain including a protein pore and an antibody-mimetic binder having a binding affinity for a target analyte tethered to the protein pore positioned in the membrane. The protein pore may comprise a monomeric β-barrel scaffold. The monomeric β-barrel scaffold may be a monomeric β-barrel scaffold of a tFhuA protein. The sensor may further comprise an FN3 monobody. The FN3 monobody may be tethered to the protein pore by a (GGS)2 tether. The (GGS)2 tether may be coupled to an N-terminus of the monomeric β-barrel scaffold of the tFhuA protein. The antibody-mimetic binder includes a tenth fibronectin type-III domain.
In another aspect, the present invention may be a method of detecting a target analyte. The method may include the step of providing a single polypeptide chain including a protein pore and an antibody-mimetic binder having a binding affinity for the target analyte. The method may further include the step of reconstituting the single polypeptide chain in a lipid membrane having a predetermined membrane potential to form a sensor. The method may additionally comprise the step of exposing the sensor to a solution that potentially contains the target analyte. The method further comprises measuring any change in the membrane potential. The protein pore may comprise a monomeric β-barrel scaffold. The monomeric β-barrel scaffold may be a monomeric β-barrel scaffold of a tFhuA protein. The sensor may further comprise an FN3 monobody. The FN3 monobody may be tethered to the protein pore by a (GGS)2 tether. The (GGS)2 tether may be coupled to an N-terminus of the monomeric β-barrel scaffold of the tFhuA protein. The antibody-mimetic binder includes a tenth fibronectin type-III domain.
The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:
Referring to the figures, wherein like numerals refer to like parts throughout, there is seen in
As explained above, the present invention was implemented by creating three sensors 10 using FN3SUMO, Mb4, and Adnectin1 monobodies as binders against hSUMO1, WDR5, and the ectodomain of EGFR, respectively. These monobody-based sensors are denoted by FN3SUMO-tFhuA, Mb4-tFhuA, and Adnectin1-tFhuA, respectively (
An artificial intelligence approach was used to elucidate the overall three-dimensional conformation of a folded protein using its amino acid sequence. The most suited structural model for FN3-tFhuA was reached when predicted Local Distance Difference Test (pLDDT), a confidence score per residue, was between 80 and 100 for most residues (
An inspection of all sensors at a transmembrane potential of +40 mV revealed a relatively quiet single-channel electrical current recorded with FN3 SUMO-tFhuA and Mb4-tFhuA, and a slightly noisy signal acquired with Adnectin1-tFhuA (
Values represent mean±s.d. from n=3 distinct experiments.
Real-Time Protein Detection Using FN3SUMO-tFhuA
FN3SUMO-tFhuA was first functionally reconstituted into a membrane at an applied transmembrane potential of +40 mV. The presence of hSUMO1 in the cis compartment at nanomolar concentrations produced frequent current blockades (
Moreover, hSUMO1-captured events were noted in a concentration-dependent manner when hSUMO1 was added to the cis compartment (
Next, detailed statistical analyses were performed of both the hSUMO1-released and hSUMO1-captured durations, whose mean values were denoted by τon and τoff, respectively. The maximum likelihood method and logarithm likelihood ratio (LLR) tests were employed to determine the most accurate distribution model of these time constants. Durations of hSUMO1-released and hSUMO1-captured events showed a single-exponential distribution in the form of a single peak in a semilogarithmic representation (
Values are mean±s.d. from n=3 independent experiments. The applied transmembrane potential was +40 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, 0.5 mM TCEP, pH 8.0.
Here, the association rate constants, kon, were consistent for all [hSUMO1] values (Table 4). In addition, the frequency of hSUMO1-captured events, f, where f=1/τon, was proportional to [hSUMO1] in a ratio 1:1 (
Values are mean±s.d. from n=3 independent experiments. The applied transmembrane potential was +40 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, 0.5 mM TCEP, pH 8.0.
Values are provided as mean±s.e.m. The other experimental conditions were the same as those stated in Methods.
Detection of a Chromatin-Associated Protein Hub Using Mb4-tFhuA
The same approach was used to detect WDR5 using a functionally reconstituted Mb4-tFhuA sensor into a lipid bilayer. When added to the cis compartment at nanomolar concentrations, WDR5 produced frequent current blockades (
Values are mean±s.d. from n=3 independent experiments. The applied transmembrane potential was +40 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, 1 mM TCEP, pH 8.
Values are mean±s.d. from n=3 independent experiments. The applied transmembrane potential was +40 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, 1 mM TCEP, pH 8.
Orthogonal measurements were found to prove the rapid association and dissociation kinetics of WDR5-Mb4 interactions. To validate the fast kinetics recorded with the Mb4-tFhuA sensor, an orthogonal experiment using biolayer interferometry (BLI) was performed. Mb4-tFhuA-containing micelles were immobilized onto the BLI sensor surface using biotin-streptavidin chemistry (Methods;
Values are mean±s.e.m. The other experimental conditions were the same as those stated in Methods.
Detection of EGFR biomarker using an Adnectin1-tFhuA. The ectodomain of EGFR is proteolytically released into the bloodstream, allowing this biomarker to be used for screening, diagnosis, and disease progression. Hence, we employed the Adnectin-1 binder against the ectodomain of EGFR. The main benefit of Adnectin-1 is its high affinity with EGFR with a KD of 2 nM. Here, we show real-time detection of EGFR using an Adnectin1-tFhuA sensor. We noted that Adnectin1-tFhuA sensor exhibits some current noise at +40 mV (
Moreover, the relative position and probability of these peaks, for low-amplitude and large-amplitude blockades, were independent of EGFR concentration, [EGFR] (Table 9). EGFR-released (Oon) and EGFR-captured (Ooff) durations followed single-peak and double-peak event distributions (
Values of the normalized current blockades are mean±s.d. from n=3 independent experiments.
Values are mean±s.d. from n=3 independent experiments. P1, and P2 are the probabilities of the short- and long-lived EGFR-captured events (Table 9), respectively. The applied transmembrane potential was +20 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, pH 8.0. These events were differentiated by the EGFR-captured duration. Individual experimental values were derived using event histograms in ClampFit (Axon) and fits in a semilogarithmic representation. The maximum likelihood method and logarithm likelihood ratio (LLR) tests were used for all fits to determine the best multi-exponential distribution model (Methods).
Values are mean±s.d. from n=3 independent experiments. The applied transmembrane potential was +20 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, pH 8.0. All histogram fittings were conducted using a semilogarithmic representation. The maximum likelihood method4, 5 and logarithm likelihood ratio (LLR) tests6-8 were used for all fits to determine the best multi-exponential distribution model (Methods).
Values are mean±s.d. from n=3 independent experiments. The applied transmembrane potential was +20 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, pH 8.0. Subscripts “i” 1 and 2 stand for short- and long-lived EGFR capture events, respectively. τon are mean values of the single-exponential distributions of EGFR-released duration histograms. τon-1=τon/P1, where P1 is the event probability of the short-lived events for each experiment. τon-2=τon/P2, where P2 is the event probability of the long-lived events for each experiment. The mean values of those probabilities are listed in Table 8. All histogram fits were conducted using a semilogarithmic representation. The maximum likelihood method4, 5 and logarithm likelihood ratio (LLR)6, 7, 9 tests were used for all fits to determine the best multi-exponential distribution model (Methods).
Furthermore, the frequency of EGFR-captured was amplified by increasing the EGFR concentration, [EGFR] (
Values are mean±s.d. from n=3 independent experiments. The applied transmembrane potential was +20 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, pH 8.0.
Values are mean±s.d. from n=3 independent experiments. The applied transmembrane potential was +20 mV. The buffer solution contained 300 mM KCl, 10 mM Tris-HCl, pH 8.0.
The EGFR structure in the EGFR/EGF complex (1NQL.pdb) is mostly similar that of EGFR in the EGFR-Adnectin1 complex (3QWQ.pdb) and is believed to be an inactive form of the receptor (
Next, an investigation was performed to determine whether these reversible current transitions may also involve transitions taking place between the two EGFR-captured substates, “1” and “2”. Hence, a related question is whether a kinetic model including interconversion transitions between these capture substates would more accurately reflect experimentally determined rate constants. An interconversion-dependent kinetic model was then developed, encompassing two supplementary rate constants between EGFR-captured substates, k12 and k21 (Table 16,
Values are mean±s.e.m. The other experimental conditions were the same as those stated in Methods. The association and dissociation rate constants and the equilibrium dissociation constant, KD. kon-1 and kon-2 values are the slopes of the linear fits in
Raw single-channel traces were analyzed event using MATLAB (MathWorks, Natick, MA). The EGFR concentration was 20 nM. Values were provided as mean±s.d. (n=3), where n is the number of independently reconstituted nanopores. The association rate constants kon-1 and kon-2 obtained by this model were the same as those determined by the interconversion-independent kinetic model. The interconversion-independent and interconversion-dependent kinetic models are illustrated in
Single-molecule detection of a protein biomarker in a biofluid.
These sensors were challenged in the presence of 5% fetal bovine serum (FBS) to examine the stability of this system in a challenging environment and the ability to distinguish analyte-capture events from other nonspecific transitions of the serum constituents. The serum threshold for the soluble EGFR ectodomain level is 45 ng/ml (˜112 nM). The tumor state can be evaluated at EGFR levels significantly exceeding this threshold.
Values are mean±s.d. from n=3 independent experiments. P1 and P2 are the probabilities of the short- and long-lived EGFR-captured events, respectively. The other experimental conditions were the same as those stated in Methods. These events were differentiated by the EGFR-captured duration. Individual experimental values were derived using event-list histograms in ClampFit (Axon) and their fits in a semilogarithmic representation. The maximum likelihood method and logarithm likelihood ratio (LLR) tests were used for all fits to determine the best multi-exponential distribution model (Methods).
Values in are mean±s.d. (− is the absence of FBS from n=3 independent experiments and + is the presence of FBS from n=3 independent experiments). The applied transmembrane potential was +20 mV. The buffer solution contained 20 nM EGFR, 300 mM KCl, 10 mM Tris-HCl, pH 8.0.
Values are mean±s.d. (− is the absence of FBS from n=3 independent experiments and + is the presence of FBS from n=3 independent experiments). The applied transmembrane potential was +20 mV. The buffer solution contained 20 nM EGFR, 300 mM KCl, 10 mM Tris-HCl, pH 8.0.
Distinctive Outcomes with Monobody-Based Sensors
The present invention provides a detailed signature analysis of single-molecule protein detection of three analytes using three nanopore sensors that share a common modular architecture, but they differ by their binding surface (
Capture durations represent mean±s.e.m using the fits in
Validation of the Monobody-Based Sensors
To validate these monobody-based sensors, we examined the binding affinity of detergent-refolded sensors with their cognate protein analytes using steady-state FP anisotropy. If the labeled protein analyte interacts with the corresponding monobody-containing nanopore sensor, then its tumbling rate (e.g., the coefficient of rotational diffusion) would decrease, increasing the FP anisotropy. In accord with our expectation, the FP anisotropy substantially increased at elevated sensor concentrations (
Advantages of this Class of Protein Sensors and its Implications in Nanobiotechnology
The new class of nanopore sensors made of a single-polypeptide chain protein according to the present invention circumvents the necessity of tedious purification steps, otherwise needed for multimeric nanopore sensors. The overall architecture of the sensors can be maintained while changing the binding interface of the antibody-mimetic binder. This way, such an approach substantially expands the utility of these sensing elements for numerous protein biomarkers while preserving their high specificity and sensitivity. This critical benefit is facilitated by the genetically encoded nature of these sensors so that they can create combinatorial libraries of tethered binders. In addition, there is no fundamental limitation or technical challenge in replacing the monobody with another small protein scaffold binder, such as an affibody or an anticalin. Furthermore, the main advantages of using antibody-mimetic proteins include strong binding affinities with different epitopes, straightforward expression and purification procedures, lack of disulfide bonds, and high thermodynamic stability. Fortuitously, in the current sensor configuration, monobodies orient about 90° with respect to the central axis of tFhuA, allowing direct electrical detection of analyte bindings without needing an adapter. Because most antibody-mimetic scaffolds are developed against the native binding sites of proteins, our sensors can be potentially utilized for the screening of specific libraries of small-molecule inhibitors. These sensors can operate in challenging biofluids at clinically relevant concentration ranges and with an extended time bandwidth. In this process, the biomarker-induced events are unambiguously distinguished from other nonspecific bindings of biofluid constituents. Therefore, with further development these synthetic nanopore sensors can be integrated into nanofluidic devices and coupled with high-throughput technologies for biomarker profiling in biomedical diagnostics.
Methods
Synthetic Gene Construction.
Three derivatives of wild-type fibronectin type-III (10FN3) were used for the development of these sensors. The cDNA sequences of these 10fn3 genes, namely fn3sumo, mb4, and adnectin 1, were fused to the 5′ end of the tfhua gene via a (GGS)2-encoding linker by a restriction-free cloning method. The cDNA sequences of Mb4 and Adnectin1 were synthesized by Eurofins Genomics (Louisville, KY) and Integrated DNA Technologies (IDT, Coralville, Iowa), respectively. The construction of the fn3sumo gene was made based on ySMB9. The cDNA sequence of all three fibronectin derivatives was first amplified using Q5 high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA) from their respective template DNA. PCR products were separated on 1% agarose gel and purified using a Gel extraction kit (Promega, CA). Sequences of forward and reverse primers are listed in Table 21. Amplified products of fn3sumo and mb4 genes were then fused to the 5′ end of tfhua cloned in pPR-IBA1 plasmid (IBA, Goettingen, Germany). The adnectin1 gene was joined at the 5′ end of the tfhua gene in pET28a (EMD Millipore, Burlington, MA). The pET28-tFhuA plasmid was constructed by inserting the gene between BamHI and XhoI restriction sites after amplification with forward and reverse primers of tFhuA (Table 21). All the gene sequences were verified by sequencing (MCLab, San Francisco, CA). The pET11a-hSUMO1 was kindly provided by Fauke Mechior (Addgene plasmid #53138).
Protein Expression and Purification
For the expression of FN3SUMO-tFhuA, Mb4-tFhuA, and Adnectin1-tFhuA, the plasmids mentioned above were transformed into E. coli BL21(DE3) cells. These monobody-containing protein nanopores were purified as previously described. The protein purity was validated by SDS-PAGE analysis (
In the case of hSUMO1, BL21(DE3) cells were transformed with pET11a-hSUMO1 and grown in Luria-Bertani (LB) medium at 37° C. until OD600 reached a value of ˜0.5. Then, the temperature was changed to 20° C. Expression was initiated by inducing the cells with 250 μM IPTG. After induction, the cells were cultured for ˜18 h at 20° C. Cells were then centrifuged at 3,700 g for 30 min at 4° C., followed by their resuspension in 50 mM Tris-HCl, 50 mM NaCl, and pH 8.0. The lysozyme was added to the suspended cells and incubated on ice for 15 min, and cell lysis was accomplished using sonication (30 s on, 60 s off×4 times). The cell lysate was centrifuged at 108,500 g for 30 min at 4° C. to separate the insoluble pellet and supernatant. The supernatant was collected and filtered using a 0.22 μm filter. The supernatant was loaded onto Q-Sepharose column (Cytiva, Marlborough, MA), which was washed with 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, and eluted with 50 mM Tris-HCl, 1 M NaCl, pH 8.0 in a gradient manner. The desired fractions were collected, dialyzed, and concentrated. Furthermore, the protein sample was loaded on an S75 gel-filtration column (GE Healthcare, Chicago, IL). Pure fractions were collected and dialyzed against 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, and 0.5 mM TCEP overnight at 4° C. Purification of WDR5 was done as described previously.
For the purification of the ectodomain of epidermal growth factor receptor (EGFR), Expi293F cells (Thermo Fischer Scientific) were seeded at 106 cells/ml density in 1 L of Dynamis growth medium (Gibco) 24 h before the transfection and supplemented with Tryptone/Glucose. For the sake of simplicity, we name this EGFR throughout this article. The culture was transfected with 2 μg/mL of the pCMV_EGFR plasmid containing the signal peptide with 3.75×polyethylenimine (PEI). Transfected cells were cultured for five days, and the protein was allowed to excrete from the cells. Five days post-transfection, the culture was pelleted, and the supernatant was filtered. The sample was loaded onto an immobilized metal-affinity column (1 mL, HIStrap HP column, GE Healthcare), which was washed with 50 mM sodium phosphate (NaPi) (pH 8.0), 300 mM NaCl, 20 mM imidazole. The protein was eluted using 50 mM NaPi (pH 8.0), 300 mM NaCl, and 500 mM imidazole. Peak fractions were collected and confirmed by SDS-PAGE (
Protein Refolding
The purified FN3SUMO-tFhuA, Mb4-tFhuA, and Adnectin1-tFhuA were adjusted to a final concentration of ˜10 μM. Next, n-dodecyl-β-d-maltopyranoside (DDM) was added to denatured samples to a final concentration of 1% (w/v). The protein samples were immediately dialyzed against the buffer containing 200 mM KCl, 20 mM Tris-HCl, pH 8, at 4° C. for 96 h. The dialysis solution was replaced at 24-h intervals. These refolded protein samples were centrifuged to eliminate any protein precipitations, and the supernatant was used as the running sample for single-channel electrical recordings. Protein concentrations were determined by their molar absorptivity at a wavelength of 280 nm.
Single-Channel Electrical Recordings
Electrical detection of protein ligands at single-molecule precision was conducted using planar lipid bilayers. The two halves of the chamber were divided by a 25 μm-thick Teflon septum (Goodfellow Corporation, Malvern, PA). A planar lipid bilayer was made of 1,2-diphytanoyl-sn-glycero-phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) across an ˜100 μm-diameter aperture of the Teflon septum. For all experiments, the buffer solution contained 300 mM KCl, 10 mM Tris-HCl, and pH 8.0. In addition, this buffer included 0, 0.5, and 1 mM TCEP in experiments with EGFR, hSUMO1, and WDR5, respectively. The nanopore protein samples (final concentration, 0.5-1.5 ng/μ1) and analytes were added to the cis compartment, which was grounded. Single-channel electrical currents were acquired using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). The applied transmembrane potential was +40 mV, unless otherwise stated. The electrical signal was sampled at 50 kHz using a low-noise acquisition system (Model Digidata 1440 A; Axon Instruments). A low-pass Bessel filter (Model 900; Frequency Devices, Ottawa, IL) was further employed for signal filtering at 10 kHz. For the data processing and analysis, the electrical traces were digitally filtered with a low-pass 8-pole Bessel filter at 3 kHz, unless otherwise stated. All single-channel electrical recordings were acquired at a temperature of 24±1° C.
EGFR Detection in a Heterogeneous Solution
Fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Pittsburgh, PA) was sterile-filtered using a 0.2-μm filter and stored at −80° C. in aliquots for long-term use. A fresh aliquot was defrosted on ice and incubated for at least 30 minutes at room temperature before adding to the chamber. Single-channel electrical traces were recorded in the presence of FBS at a final concentration of 5%.
Biolayer Interferometry (BLI) Assay Using Immobilized Proteomicelles
These experiments were conducted using an Octet Red384 instrument (FortéBio, Fremont, CA) at 24° C. For BLI experiments, a site specific insertion of cysteine at position 287 was achieved in the long loop of Mb4-tFhuA by site directed mutagenesis (Q5 mutagenesis kit, New England Biolabs). This cysteine-containing Mb4-tFhuA was expressed and purified as described above except for the presence of reducing agent. Cys287 was biotinylated using maleimide. A flexible (PEG)11 linker was used between the biotin and maleimide group. In this way, there was a satisfactory distance between Mb4 and the surface of BLI sensor. The BLI running buffer contained 300 mM KCl, 20 mM Tris-HCl, 1 mM TCEP, 1% DDM, 1 mg/ml bovine serum albumin (BSA), pH 8.0. It was used to soak streptavidin (SA) sensors for 30 min. The 50 nM Mb4-tFhuA_Cys287-(PEG)11-Biotinyl was loaded onto the sensors for 2.5 min via biotin-streptavidin chemistry. By dipping the sensors in a protein-free solution for 6 minutes, the unattached Mb4-tFhuA_Cys287 was washed away. The association process was examined using various concentrations of WDR5, ranging from 1.5 μM to 6 μM. To inspect the dissociation phase, the BLI sensors were dipped in a WDR5-free running buffer. For all WDR5 concentrations, the Mb4-tFhuA_Cys287-free BLI sensors were run in parallel as controls. The baseline and drift in the sensorgrams were subtracted using these controls. The FortéBio Octet data analysis software (ForteBio) was used for the sensorgram analysis.
Steady-State Fluorescence Polarization (FP) Measurements
hSUMO1 and WDR5 were labeled with fluorescein and rhodamine, respectively, at pH9.0 by primary amine chemistry. These labeled proteins were added to the well at a final concentration of 50 nM. Steady-state fluorescence polarization (FP) anisotropy assays were conducted in triplicate with an 18-point serial dilution of FN3SUMO-tFhuA, Mb4-tFhuA, or unmodified tFhuA, against a fixed concentration of labeled proteins on black 96-well plates. All steady-state FP measurements were recorded using a SpectraMax i3x plate reader (Molecular Devices, San Jose, CA) at 0 min and after a one-hour incubation at room temperature in the dark. The resulting dose-response data were averaged and fitted using a logistic regression to obtain the dissociation constant (KD) for each interaction.
Statistical Analysis
pClamp 10.7 (Axon Instruments) was used for the data acquisition and analysis. Capture and release events were collected using the single-channel event search in ClampFit 10.7 (Axon Instruments), and figures were prepared by Origin 9.7 (OriginLab, Northampton, MA). The probability distribution function (PDF) was generated using a kinetic rate matrix, and the kinetic rates were determined by fitting the data using the maximum likelihood method. To evaluate the results of multiple models and select the number of statistically significant peaks that are best matched to the data, a logarithm likelihood ratio (LLR) test was performed. At a confidence number of 0.95, a single-exponential fit was the best model for the release and capture durations of hSUMO1 and WDR5. For EGFR, a two-exponential fit was the best model for the capture durations.
Molecular Graphics
All cartoons showing molecular graphics were prepared using the PyMOL 2 (Version 2.4.0; Schrödinger, LLC) and Chimera X (Version 1.4; University of California at San Francisco).
In another example of sensor 10 as seen in
The present application claims priority to U.S. Provisional Application No. 63/409,906, filed on Sep. 26, 2022.
This invention was made with government support under Grant GM088403 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
Number | Date | Country | |
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63409906 | Sep 2022 | US |