human full-length, mneongreen tagged ephb6 sequences  (GenScript corporation)

Journal: Pharmaceuticals

Article Title: FRET Assays for the Identification of C. albicans HSP90-Sba1 and Human HSP90α-p23 Binding Inhibitors

doi: 10.3390/ph17040516

Figure Lengend Snippet: FRET assay setup for C. albicans HSP90–Sba1 or human HSP90α–p23 inhibitor screening. The FRET assay setup is explained for C. albicans HSP90–Sba1 binding. The corresponding assay setup for the human complex formation is analogous. ( 1 ) HSP90-mNeonGreen fusion proteins form homodimers that are dimerized at the C-terminal domain. HSP90-mNeonGreen adopts an open conformation in the absence of ATP. ( 2 ) Upon ATP binding, HSP90-mNeonGreen can progress to an N-terminally dimerized state. ( 3 ) The ATP-bound twisted configuration of HSP90-mNeonGreen enables the binding of Sba1-mScarlet-I to HSP90-mNeonGreen. Two Sba1 molecules can bind per HSP90 dimer. During this complex formation, the donor and acceptor come into close contact. This state allows FRET between the donor fluorescent protein mNeonGreen and the acceptor fluorescent protein mScarlet-I, resulting in an increase in FRET emission. Concomitantly, as energy is transferred from the donor fluorophore to the acceptor fluorophore via FRET, the donor fluorescence decreases compared to non-binding samples. Binding of Sba1 to HSP90 stabilizes the ATP-bound conformation of HSP90 leading to a deceleration of HSP90 ATPase function and its conformational cycle progression. Subsequent to ATP hydrolysis, HSP90 adopts the open conformation again ( 1 ), and Sba1 dissociates from the complex. ( 4 ) When an inhibitor of HSP90–Sba1 binding is added to this setup, no FRET occurs, resulting in a low sensitized emission as well as no reduction in fluorescence emission. Since the ATP-bound conformation of HSP90 is a prerequisite for HSP90–Sba1 binding, the assay is suitable for the identification of HSP90–Sba1 protein–protein interaction (PPI) inhibitors, as well as for the identification of ATP-competitive HSP90 inhibitors. HSP90 monomers are depicted in dark gray and gray. Fluorescent protein mNeonGreen is depicted in lime. Sba1 is shown in beige. Fluorescent protein mScarlet-I is colored red. Homology models of C. albicans HSP90 open and closed conformations (based on PDB IDs 2IOQ and 2CG9, respectively) and HSP90-Sba1 complex (based on PDB ID 2CG9) were created using the Swiss Model server . The figure was created with ChimeraX 1.7.1 .

Article Snippet: The incorporation of the mNeonGreen sequence was performed by In-Fusion Cloning (Clontech Laboratories, Takara, Saint-Germain-en-Laye, France).

Techniques: Binding Assay, Fluorescence

Journal: Pharmaceuticals

Article Title: FRET Assays for the Identification of C. albicans HSP90-Sba1 and Human HSP90α-p23 Binding Inhibitors

doi: 10.3390/ph17040516

Figure Lengend Snippet: Specificity and equilibrium dissociation constant ( K d ) determination of C. albicans HSP90–Sba1 and human HSP90α–p23 binding via FRET. ( A , C ) All donor concentrations were kept constant at 1 µM. The acceptor was varied in a concentration range of 0–2250 nM (lowest non-zero concentration 53 nM). ( A ) Specificity of C. albicans HSP90-mNeonGreen binding to Sba1-mScarlet-I in comparison with donor control and acceptor control. When omitting ATP from the reaction buffer (grey down-pointing triangles), HSP90-mNeonGreen–Sba1-mScarlet-I binding is abrogated. mNeonGreen (green squares) and mScarlet-I (red up-pointing triangles) show a linear, unspecific rise in FRET emission. ( B ) The C. albicans HSP90E36A-mNeonGreen concentration was kept constant at 200 nM. Sba1-mScarlet-I concentration was varied from 0–3000 nM (lowest non-zero concentration 10 nM). The samples were incubated at 37 °C for 3 h to ensure equilibrium. The determined K d value was 100 nM (PCI: 80–120 nM, ACI: 100–140 nM). ( C ) Shown are the results for the human homologous complex formation of HSP90α–p23. ( D ) The experiment for determining the K d of human HSP90αE47A-mNeonGreen–p23-mScarlet-I binding was performed analogously to ( B ) except for incubating at 37 °C for 3 h. The K d value was 210 nM (PCI: 170–250 nM, ACI: 180–260 nM). Em FRET : FRET emission, R.F.U.: relative fluorescence units, PCI: precision confidence interval, ACI: accurate confidence interval. Both PCI and ACI were calculated at a 95.5% confidence level. Error bars represent the standard deviation. Experiments with varying incubation times to test for equilibration are included in the . Reports for ACI determination are included in the .

Article Snippet: The incorporation of the mNeonGreen sequence was performed by In-Fusion Cloning (Clontech Laboratories, Takara, Saint-Germain-en-Laye, France).

Techniques: Binding Assay, Concentration Assay, Comparison, Incubation, Fluorescence, Standard Deviation

Journal: Pharmaceuticals

Article Title: FRET Assays for the Identification of C. albicans HSP90-Sba1 and Human HSP90α-p23 Binding Inhibitors

doi: 10.3390/ph17040516

Figure Lengend Snippet: Characterization of model C. albicans HSP90–Sba1 and human HSP90α–p23 binding inhibitors via FRET. ( A ) Sba1 selectively competes with Sba1-mScarlet-I for binding to HSP90-mNeonGreen. HSP90-mNeonGreen (1 µM) and Sba1-mScarlet-I (2 µM) were incubated in reaction buffer containing 5 mM ATP. The addition of Sba1 (20 µM) showed a significant reduction ( p < 0.001, depicted as ***) in the observed Em FRET in comparison to the untreated control (UC). When adding bovine serum albumin (BSA) (20 µM) to the aforementioned constant concentrations of HSP90-mNeonGreen/Sba1-mScarlet-I, there was no significant (n.s.) reduction in Em FRET . ( B ) p23 selectively competes with p23-mScarlet-I for binding to HSP90α-mNeonGreen. The experiment was performed analogously to ( A ). ( C ) Sba1 competes with Sba1-mScarlet-I for binding to HSP90-mNeonGreen in a dose-dependent manner. HSP90-mNeonGreen (1 µM) and Sba1-mScarlet-I (1 µM) were incubated in reaction buffer containing 5 mM ATP. The determined IC 50 is 1950 ± 230 nM. ( D ) p23 competes with p23-mScarlet-I for binding to HSP90α-mNeonGreen in a dose-dependent manner. The determined IC 50 is 1420 ± 190 nM. The experiment was performed analogously to ( C ). ( E ) Small molecule HSP90 inhibitor geldanamycin (GA) disrupts the binding of Sba1-mScarlet-I to the ATP-hydrolysis-defective mutant HSP90E36A-mNeonGreen in a dose-dependent manner. HSP90E36A-mNeonGreen (1 µM) and Sba1-mScarlet-I (2 µM) were incubated in reaction buffer containing 5 mM ATP. The geldanamycin concentration was varied in a range of 0–400 µM (lowest non-zero concentration 1.4 µM). The determined IC 50 is 60 ± 10 µM. ( F ) GA disrupts the binding of p23-mScarlet-I and the ATP-hydrolysis-defective mutant HSP90αE47A-mNeonGreen in a dose-dependent manner. HSP90αE47A-mNeonGreen (1 µM) and p23-mScarlet-I (2 µM) were incubated at 37 °C for 15 min in reaction buffer containing 5 mM ATP. The determined IC 50 is 17 ± 3 µM. ( G ) ATP concentration shows a strong influence on HSP90E36A-mNeonGreen–Sba1-mScarlet-I binding. HSP90E36A-mNeonGreen (1 µM) was incubated with Sba1-mScarlet-I (2 µM) in reaction buffer. ATP was varied in a concentration range of 0–12,500 µM (lowest non-zero concentration 3 µM). The EC 50 is 220 ± 40 µM. ( H ) ATP concentration shows a strong influence on HSP90αE47A-mNeonGreen–p23-mScarlet-I binding. HSP90αE47A-mNeonGreen (1 µM) was incubated with p23-mScarlet-I (2 µM) in reaction buffer. The EC 50 is 210 ± 30 µM. Error bars represent the standard deviation. Given error is the error of the fit. Em FRET [R.F.U.]: FRET emission in relative fluorescence units. ( E – H ) Maximum Em FRET was normalized to 1 to represent the full binding of HSP90 and co-chaperone.

Article Snippet: The incorporation of the mNeonGreen sequence was performed by In-Fusion Cloning (Clontech Laboratories, Takara, Saint-Germain-en-Laye, France).

Techniques: Binding Assay, Incubation, Comparison, Mutagenesis, Concentration Assay, Standard Deviation, Fluorescence

Journal: Pharmaceuticals

Article Title: FRET Assays for the Identification of C. albicans HSP90-Sba1 and Human HSP90α-p23 Binding Inhibitors

doi: 10.3390/ph17040516

Figure Lengend Snippet: Categorization of screening assay quality and validation. ( A ) Binding control samples containing 3 mM ATP (blue circles) show high FRET emission ( Em FRET ) as well as a lower donor fluorescence ( FL DD ) compared to the non-binding control containing no ATP (red squares). C. albicans HSP90-mNeonGreen (1 µM) and Sba1-mScarlet-I (2 µM) in reaction buffer with 1% DMSO containing either 3 mM ATP or no ATP were incubated for 15 min at 30 °C prior to measurement. ( B ) Calculation of the quotient of FRET emission and donor emission ( Em FRET / FL DD ) results in robust separation and a Z ′ factor of 0.58. ( C ) The screening assay can identify HSP90-Sba1 binding inhibitors with a high degree of confidence. HSP90-mNeonGreen (1 µM) and Sba1-mScarlet-I (2 µM) in reaction buffer, 3 mM ATP and 1% DMSO were incubated with various literature-described HSP90 inhibitors at concentrations of 10 or 100 µM for 15 min at 30 °C prior to measurement. When the hit threshold is defined as 3 SDs of the binding control mean (blue circles), ATP-competitive inhibitors of HSP90 geldanamycin, radicicol, luminespib (NVP-AUY922), SNX-5422 and BIIB021 are reliably identified as disrupting HSP90-Sba1 binding. Non-ATP competitive HSP90 inhibitors silibinin, deguelin and withaferin A do not show an effect on HSP90–Sba1 binding. Furthermore, Sba1 (20 µM) is also identified as disrupting HSP90-mNeonGreen–Sba1-mScarlet-I binding. ( D – F ) The assay conditions for human HSP90α-mNeonGreen–p23-mScarlet-I binding inhibitor identification are analogous to the C. albicans assay, with the exception that samples were incubated for 15 min at 37 °C prior to measurement. ( E ) For the human HSP90–p23 inhibitor screening assay, a Z ′ factor of 0.32 was calculated. This classifies the assay as a double assay, indicating that compounds screened for HSP90α–p23 binding inhibition should be screened in duplicates. ( B , C , E , F ) Solid lines represent means of each data set (binding control or non-binding control). Dashed lines represent 3 standard deviations (SDs) from the respective mean.

Article Snippet: The incorporation of the mNeonGreen sequence was performed by In-Fusion Cloning (Clontech Laboratories, Takara, Saint-Germain-en-Laye, France).

Techniques: Screening Assay, Binding Assay, Fluorescence, Incubation, Inhibition