Journal: Cell Chemical Biology

Article Title: Differences in the Conformational Energy Landscape of CDK1 and CDK2 Suggest a Mechanism for Achieving Selective CDK Inhibition

doi: 10.1016/j.chembiol.2018.10.015

Figure Lengend Snippet: Inhibitor Binding to CDK1 and CDK2 (A and B) Isothermal titration calorimetry (ITC) thermograms to assess AZD5438 binding to CDK1-cyclin B and CDK2-cyclin A (A) and cyclin-free CDK1 and CDK2 (B). For each sample, CDK1 and CDK2 were phosphorylated (on T161 or T160, respectively). AZD5438 shows reduced binding to cyclin-free CDK1 compared with CDK1-cyclin (B). (C) Surface plasmon resonance (SPR) studies to determine the binding of AZD5438 to CDK1 and CDK2. Unphosphorylated CDK1 and CDK2 as GST fusions were immobilized on the SPR chip via anti-GST antibody coupling. Accompanying sets of ITC thermograms and SPR traces that evaluate Dinaciclib, SU9516, Alvocidib, and CGP74514A binding are presented in Figure S2 . (D) Bar chart to compare the fold difference in binding affinity between cyclin-free CDK1 and CDK2 and their cyclin-associated forms. CDK1:CDK1-cyclin B and CDK2:CDK2-cyclin A ratios are shown in black and gray, respectively. ITC experiments conducted in the presence of Cks2 are presented in Figure S3 . (E) ITC-derived energetic experimental data (ΔH, -TΔS, and ΔG) for the binding of AZD5438 to CDK1 and CDK2 and their respective cognate cyclins.

Article Snippet: Individual protein samples were exchanged into ITC buffer (40 mM HEPES, 500 mM NaCl, 0.25 mM TCEP, pH 8.0) at 4°C using a Hitrap desalting column (GE Healthcare Life Sciences, Chicago, IL, USA) (i.e. CDK1 and cyclin B were not combined to form a complex prior to buffer exchange).

Techniques: Binding Assay, Isothermal Titration Calorimetry, SPR Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes *

doi: 10.1074/jbc.M110.148262

Figure Lengend Snippet: A, hypothetical structural organization of Oxa1L in the inner membrane (IM) is shown. Oxa1L is composed of an N-terminal domain in the intermembrane space (IMS), a transmembrane domain (TMD) with five transmembrane helices, and a C-terminal domain located in the matrix. B, the primary sequence of the Oxa1L-CTT expressed and purified from E. coli is shown. Regions predicted to be α-helical by the secondary structure prediction programs on Biology Workbench using the PELE collection of programs are underlined. The methionine (M) at the beginning of the sequence and the LEHis6 at the C terminus of the sequence are from the vector. C, the Rosetta structure prediction protocol was used to generate a model of Oxa1L-CTT (26). The structure shown is the lowest free-energy structure and is displayed using PyMOL. D, prediction of coiled-coil formation is shown. The coiled-coil structure was predicted using the COILS program with two different windows. The figure shows the comparison of coiled-coil-forming tendency in a 14-residue window of the C-terminal tail of yeast, human, bovine, and mouse Oxa1. The amino acid listed as zero in the figure corresponds to residues 317, 334, 334, and 330 residues in full-length yeast, human, bovine, and mouse Oxa1, respectively.

Article Snippet: The cDNA clone of human Oxa1L was obtained from American Type Culture Collection (ATCC number 10961183, IMAGE 40017377).

Techniques: Sequencing, Purification, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes *

doi: 10.1074/jbc.M110.148262

Figure Lengend Snippet: Effect of protein concentration, salt, and TFE on the secondary structure of Oxa1L-CTT determined by CD. A, shown is the effect of Oxa1L-CTT concentration (0.1, 0.2, and 0.34 mg/ml) on the CD spectra. B, shown is the effect of salt concentration on the CD spectra of Oxa1L-CTT. The protein concentration was 0.1 mg/ml. C, shown is the effect of TFE concentration on the CD spectra of Oxa1L-CTT. Protein concentration was 0.2 mg/ml.

Article Snippet: The cDNA clone of human Oxa1L was obtained from American Type Culture Collection (ATCC number 10961183, IMAGE 40017377).

Techniques: Protein Concentration, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes *

doi: 10.1074/jbc.M110.148262

Figure Lengend Snippet: Oligomerization of Oxa1L-CTT. A, shown is the effect of KCl on oligomerization of Oxa1L-CTT as examined by analytical ultracentrifugation. The panels show the representative equilibrium sedimentation profiles at three different KCl concentrations at 10 mm MgCl2. The data are plotted as the absorption of Oxa1L-CTT (0.6 mg/ml) at 280 nm versus the distance from the center of the axis of rotation (radius). The lower section of each panel shows the raw data in closed circles. The lines represent the best fits. The upper panels show the residual for the corresponding given fit. The global fit of three protein concentrations (0.3, 0.6, and 0.9 mg/ml) is not shown. The data presented here were obtained at 24,000 rpm. B, detection of dimer and tetramer formation of Oxa1L-CTT by DMS cross-linking followed by Western blotting as described in “Experimental Procedures” is shown.

Article Snippet: The cDNA clone of human Oxa1L was obtained from American Type Culture Collection (ATCC number 10961183, IMAGE 40017377).

Techniques: Sedimentation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes *

doi: 10.1074/jbc.M110.148262

Figure Lengend Snippet: Interaction of the mammalian mitochondrial large ribosomal subunit (39 S) with Oxa1L-CTT analyzed by surface plasma resonance. A, shown is the RU change of Oxa1L-CTT binding to 39 S (circles) and 28 S (triangles) as a function of Oxa1L-CTT concentrations. Ribosomes (39 S and 28 S) and BSA were immobilized on a L1 sensor chip, and Oxa1L-CTT was flowed through the cell as described under “Experimental Procedures.” The RU values were recorded for each injection after 15 s of buffer exchange. The value of the RU from the cell carrying BSA has been subtracted from each value. The solid circles represent experimental data, and the line represents a sigmoidal fit. B, the salt dependence of the RU change when Oxa1L-CTT (20 μl at 0.18 μm) was used in buffer containing different KCl concentrations and injected at a flow rate of 10 μl/min, and RU values were noted from the base line after 15 s of buffer exchange.

Article Snippet: The cDNA clone of human Oxa1L was obtained from American Type Culture Collection (ATCC number 10961183, IMAGE 40017377).

Techniques: Binding Assay, Injection, Buffer Exchange

Journal: The Journal of Biological Chemistry

Article Title: Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes *

doi: 10.1074/jbc.M110.148262

Figure Lengend Snippet: Estimation of the thermodynamic parameters governing the interaction of Oxa1L-CTT with mitochondrial 55 S ribosomes using isothermal titration calorimetry. A, raw data for the binding of Oxa1L-CTT to 55 S ribosomes provided as the power output (μcal/s) as a function of time are shown. The protein concentration was 80 μm (syringe), and the 55 S ribosome concentration was 4 μm (cell). The first injection is only for the purpose of the experimental setup and is ignored for data analysis. B, shown is the amount of heat evolved at each injection normalized to the number of moles of Oxa1L-CTT injected (kcal/mol) versus the molar ratio of Oxa1L-CTT to 55 S ribosome. The solid line represents the nonlinear least squares fit for the data.

Article Snippet: The cDNA clone of human Oxa1L was obtained from American Type Culture Collection (ATCC number 10961183, IMAGE 40017377).

Techniques: Isothermal Titration Calorimetry, Binding Assay, Protein Concentration, Concentration Assay, Injection

Journal: The Journal of Biological Chemistry

Article Title: Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes *

doi: 10.1074/jbc.M110.148262

Figure Lengend Snippet: Strategy used to identify ribosomal proteins near the Oxa1L binding site. Oxa1L-CTT was incubated with 39 S subunits and cross-linked to nearby proteins using DMS as described under “Experimental Procedures.” Cross-linked complexes were purified by centrifugation through a sucrose cushion. The ribosomes were then denatured, and ribosomal proteins cross-linked to Oxa1L-CTT were recovered on Ni-NTA. Cross-linked proteins were digested with trypsin, and the proteins present were identified by LC/MS/MS.

Article Snippet: The cDNA clone of human Oxa1L was obtained from American Type Culture Collection (ATCC number 10961183, IMAGE 40017377).

Techniques: Binding Assay, Incubation, Purification, Centrifugation, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes *

doi: 10.1074/jbc.M110.148262

Figure Lengend Snippet: Peptides and ion scores of ribosomal proteins cross-linked to Oxa1L-CTT Ion scores of greater than 35 are considered significant.

Article Snippet: The cDNA clone of human Oxa1L was obtained from American Type Culture Collection (ATCC number 10961183, IMAGE 40017377).

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes *

doi: 10.1074/jbc.M110.148262

Figure Lengend Snippet: A, structural representation the putative binding site of Oxa1L mapped onto the structure of the Thermus thermophilus 50 S subunit (PDB coordinate 2WRL) using PyMOL. A, shown is a representation of the exit tunnel on bacterial ribosomes showing the traditional proteins (L22, L23, L24, and L29) near to exit tunnel of the 50 S ribosomal subunit. B, shown is a representation of the mammalian mitochondrial ribosomal proteins homologous to bacterial L13, L20, and L28 (space-filled) modeled onto the bacterial 50 S subunit. The regions of the rRNA missing in the mammalian mitochondrial ribosome have been manually removed from the coordinates for the 50 S subunit. In E. coli, L28 is almost completely covered by rRNA, but these segments of rRNA are missing in the 39 S subunit, leaving L28 more exposed to solvent. C, shown is a comparison of the sizes of bacterial and yeast L13, L20, and L28 with mitochondrial homologs. L20 is absent in yeast. The molecular weights of the MRPs are estimated after the removal of import signals predicated by MitoProt II.

Article Snippet: The cDNA clone of human Oxa1L was obtained from American Type Culture Collection (ATCC number 10961183, IMAGE 40017377).

Techniques: Binding Assay

Journal: Molecular microbiology

Article Title: Nonredundant functions of Mycobacterium tuberculosis chaperones promote survival under stress

doi: 10.1111/mmi.14615

Figure Lengend Snippet: Mtb HtpG dimerizes in vitro and interacts with Hsp90 probe geldanamycin (GA). (a) Model of Mtb HtpG (Phyre2) (Kelley et al., 2015) with relevant residues highlighted that are sites of point mutation to the UV-activatable crosslinking amino acid para-benzoyl phenyalanine (BpF or B). (b) SDS-PAGE analysis of Mtb HtpG BpF mutants at indicated residues with and without prior UV irradiation (monomer bands are indicated as “M” and dimer bands are indicated as “D”). F10, W197, and F635 consistently mediate the formation of dimer. (c) ITC analysis of Mtb HtpG (47 µM) with GA (raw data is shown on left, analysis of average of three replicates with SD shown on the right) illustrates µM affinity. (d) SDS-PAGE analysis of indicated Mtb HtpG BpF mutants without (plus 400 µM GA) and with UV irradiation (in the presence of 0, 10, 50, 100, 200, and 400 µM GA) suggests that F303 turns into the dimer interface upon binding GA, while F635 is oriented in the dimer interface regardless of ligand binding. At least three identical analyses were performed for each experiment shown

Article Snippet: ITC was carried out at 25°C in Buffer 4 using a low volume Nano ITC calorimeter (TA Instruments).

Techniques: In Vitro, Mutagenesis, SDS Page, Irradiation, Binding Assay, Ligand Binding Assay

Journal: Advanced Science

Article Title: Beyond Catalytic Therapy: Copper‐Paeonol Nanozymes Disrupt Fascin‐Mediated Actin Bundling to Suppress Tumor Growth and Metastasis

doi: 10.1002/advs.202512186

Figure Lengend Snippet: CuPaeNs bound to FSCN1. A) Bubble chart of GO enrichment analysis top 10 of cellular component after CuPaeNs treatment. B) Immunofluorescence staining of B16 cells in different groups. DAPI (blue), FSCN1 (green), and F‐actin (red). C) Molecular simulation of CuPaeNs. D) Computational model of active sites related to the potential key target of FSCN1 with paeonol, a single paeonol‐Cu 2+ unit, and CuPaeNs. The residues of the ligand‐binding domain proteins, involved in hydrogen bonds, were illustrated and marked with yellow dotted lines. E) Molecular simulation illustrating the binding affinity and detailed intermolecular binding interactions of FSCN1 with paeonol, a single Cu 2+ ‐paeonol unit, and CuPaeNs, focusing on main hydrogen bonds and hydrophobic interactions. F) Isothermal titration calorimetry result demonstrating the direct binding of CuPaeNs to FSCN1. G) CLSM images showing intracellular FSCN1 binding with CuPaeNs in B16 cells (scale bar = 20 µm) and H) responding pixel intensity plot. I) Assessment of CuPaeNs's impact on FSCN1's actin‐bundling activity using the Co‐IP assay. (J) Protein expression of FSCN1, F‐actin, and β‐actin with B16 cells in different groups, along with quantitative analysis ( n = 3). Statistically significant differences are indicated with their respective p ‐values ( *** p < 0.001).

Article Snippet: Actin‐Tracker Red‐Rhodamine, DAPI staining solution, trizol, dimethylsulfoxide (DMSO, 99.9%), apoptosis and necrosis assay kit, ATP assay kit, mitochondrial membrane potential assay kit with 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbocyanine iodide (JC‐1), β‐actin mouse monoclonal antibody (1:1000), PFKFB3 rabbit polyclonal antibody (1:1000), GAPDH mouse monoclonal antibody (1:1000), were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Immunofluorescence, Staining, Ligand Binding Assay, Binding Assay, Isothermal Titration Calorimetry, Activity Assay, Co-Immunoprecipitation Assay, Expressing

Journal: Biomolecules

Article Title: Distinct Calcium Binding and Structural Properties of Two Centrin Isoforms from Toxoplasma gondii

doi: 10.3390/biom10081142

Figure Lengend Snippet: Ca 2+ binding to TgCEN1-C and TgCEN2-C. ( A , B ) Representative Ca 2+ titration of apo-TgCEN1-C ( A ) and apo-TgCEN2-C ( B ) as monitored by ITC. Representative raw heat-power changes (upper panels) and integrated binding isotherms (bottom panels). A first injection of 0.5 μL was made and then the first data point was removed from data fitting. Curve fitting was performed by considering the two binding sites model for TgCEN1-C and the one site model for TgCEN2-C. The protein concentration was 70 μM and 100 μM, for apo-TgCEN1-C and apo-TgCEN2-C, respectively. ( C ) The downfield region of the 1 H- 15 N HSQC NMR spectra of TgCEN1-C recorded as a function of increasing Ca 2+ concentration. The molar ratio of Ca 2+ :protein in each case was 0.9, 1.5, 4, and 10 (from left to right).

Article Snippet: Decalcified ITC buffer (50 mM Tris-HCl, 150 mM KCl pH 7.5) was prepared by processing with Chelex 100 resin (BioRad, Hercules, CA, USA) [ , ].

Techniques: Binding Assay, Titration, Injection, Protein Concentration, Concentration Assay

Journal: Cell

Article Title: DNA origami signposts for identifying proteins on cell membranes by electron cryotomography

doi: 10.1016/j.cell.2021.01.033

Figure Lengend Snippet: Targeting of fluorescent protein tags by an RNA aptamer The raw data for each experiment can be found in Figure S3 . (A) Isothermal titration calorimetry (ITC) of the RNA aptamer alone binding to different fluorescent proteins. (B) Acrylamide gel-shift assay to confirm aptamer binding to fluorescent proteins. The dotted lines indicate bands corresponding to each fluorescent protein without (top line, left column) and with (bottom line, right column) aptamer. (C) Agarose gel-shift assay to confirm binding to fluorescent proteins of the aptamer incorporated into the signpost DNA origami structure. YFP fluorescence was used for detection. Shown at the top is YFP alone; at the bottom is a fusion protein of YFP and Env, the major membrane glycoprotein of murine leukemia virus. YFP and YFP-Env run as sharper bands (black arrow heads) when bound to SPOTs (right lane) than the proteins alone (left lane), as expected from decreased diffusion with SPOTs bound. YFP-Env is expected to run as two species because of the partial isomerization of a disulphide bond, which produces species with both single and triplicate copies of YFP. Some of the DNA nanostructures and associated proteins aggregate in the well (top of image). These samples were run on a single gel; the image is split to better fit figure spacing. (D) Bio-layer interferometry (BLI) was used to confirm the binding of the aptamer-functionalized origami (SPOT) to isolated fluorescent proteins (top) and to a fluorescent protein fusion to glycoprotein B (gB), a surface glycoprotein from herpes simplex virus 1 in native membrane vesicles (bottom). As observed in the other assays, SPOTs bound to the sfGFP or sfGFP-gB much more than signpost origami alone, and SPOTs specifically bound sfGFP or sfGFP-gB, not mCherry or mCherry-gB. Each experiment was run in triplicate, and the lighter region behind each measurement indicates the standard deviation across the three replicates. (E) Dynamic light scattering was used to check for aggregation in concentrated solutions of SPOTs in buffer (left) and cell culture medium (center). A small amount of aggregate was only observed in culture medium at SPOT concentrations of 200 nM, ∼20x higher than needed for cellular experiments ( Figure S6 ). The 50 nM and 10 nM samples were too dilute to observe in culture medium. The aggregation-induced control sample (gray dotted lines) was stained for electron microscopy (right) to monitor the amount of aggregation (scale bar, 200 nm). Such aggregation was not seen in normal preparations of SPOTs ( B and 2C).

Article Snippet: Fluorescent proteins and aptamer (variant without the 3′ extension for origami conjugation) were prepared in ITC buffer (PBS (ThermoFisher Scientific) + 5 mM MgCl 2 ) by dialysis using 10K Slide-A-Lyzer MINI devices (ThermoFisher Scientific) at 4°C overnight.

Techniques: Isothermal Titration Calorimetry, Binding Assay, Acrylamide Gel Assay, Shift Assay, Agarose Gel Electrophoresis, Fluorescence, Membrane, Virus, Diffusion-based Assay, Isolation, Standard Deviation, Cell Culture, Control, Staining, Electron Microscopy

Journal: Cell

Article Title: DNA origami signposts for identifying proteins on cell membranes by electron cryotomography

doi: 10.1016/j.cell.2021.01.033

Figure Lengend Snippet: Biophysical data, related to Figure 3 (A) Raw isothermal titration calorimetry data for fluorescent proteins titrated into aptamer (not linked to origami signpost). The fitted parameters are shown in the table for each binding curve. (B) We were unable to confirm binding of other aptamers using acrylamide gel shift assays. Left, an aptamer reported against hexahistidine tags ( Tsuji et al., 2009 ); Right, a reported aptamer against glycoprotein D from Herpes simplex virus I ( Yadavalli et al., 2017 ) Ci-iii. Unprocessed gel images with fluorescent detection in the GFP (i), RFP (ii), and YFP (iii) channels for gel-shift assays in B and 3C. iii Lane 3 - YFP alone. Because of its low molecular weight (∼26 kDa), we expect YFP to diffuse in the agarose gel, resulting in a band that thus appears faint and with a lower apparent motility. Lane 4 - upon addition of SPOTs to YFP, the molecular weight of the fluorescent structure would increase dramatically when they bind (∼5 MDa) so it diffuses much less, and a sharper and therefore brighter band would be seen. Lane 1 - YFP-Env has a much higher molecular weight than YFP (∼125 kDa monomer, ∼375 kDa trimer). As both forms of YFP-Env are much larger than YFP, the molecules should diffuse less in the agarose and thus appear as a more concentrated band. Additionally, there are 3 YFP on each Env trimer such that we would expect 3x the signal of YFP alone. Lane 2 - upon addition of SPOTs to YFP-Env, the trimer population of YFP-Env could bind 3 SPOTs (∼15 MDa) and the monomer population could bind one SPOT (∼5 MDa). As YFP-Env is a membrane protein produced in mammalian cells, it is glycosylated and detergent-solubilized here, both of which may impact the appearance of bands by electrophoresis. D. Example replicate buffer-subtracted and loading-normalized traces for BLI binding of unfunctionalized sign post origami (‘SPO’) or SPOTs to immobilized sfGFP-gB vesicles (i) or mCherry-gB vesicles (ii) from Figure 3 D (bottom panel). The buffer replicates (unloaded tips dipped in analyte nanostructure solutions) and average (used for subtraction) are show in iii, and the loading curves (normalized in the same way as the data) in iv .

Article Snippet: Fluorescent proteins and aptamer (variant without the 3′ extension for origami conjugation) were prepared in ITC buffer (PBS (ThermoFisher Scientific) + 5 mM MgCl 2 ) by dialysis using 10K Slide-A-Lyzer MINI devices (ThermoFisher Scientific) at 4°C overnight.

Techniques: Isothermal Titration Calorimetry, Binding Assay, Acrylamide Gel Assay, Virus, Gel Shift, Molecular Weight, Agarose Gel Electrophoresis, Membrane, Produced, Electrophoresis

Journal: Cell

Article Title: DNA origami signposts for identifying proteins on cell membranes by electron cryotomography

doi: 10.1016/j.cell.2021.01.033

Figure Lengend Snippet:

Article Snippet: Fluorescent proteins and aptamer (variant without the 3′ extension for origami conjugation) were prepared in ITC buffer (PBS (ThermoFisher Scientific) + 5 mM MgCl 2 ) by dialysis using 10K Slide-A-Lyzer MINI devices (ThermoFisher Scientific) at 4°C overnight.

Techniques: Virus, Recombinant, Transfection, Bicinchoninic Acid Protein Assay, Electron Microscopy, Expressing, Fluorescence, Ligation, Cloning, Plasmid Preparation, Software, Tomography