Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: ClpB Activation Triggers a Sequential Mode of ATP Hydrolysis (A) ClpB domain organization and monomer structure. The identity and position of mutated residues are indicated. (B) ATPase activities of ClpB wild-type (WT) and ClpB-K476C were determined in the absence and presence of 10 μM casein (± substrate). SDs are indicated; for some points, error bars are shorter than the height of the symbol and are not depicted. (C) MDH disaggregation activities of ClpB-WT and ClpB-K476C in the absence and presence of Hsp70. (D) ATPase activity of ClpB-WT and ClpB-K476C in absence and presence of casein (± substrate) as a function of ATP concentration. (E) v max of ATPase activities, derived Hill coefficient (h), and ATP concentrations at half-maximal ATPase activity ( K 0.5 ) for WT, pore 1 (Y251A), and pore 2 (Y653A) loop mutants of ClpB-WT and ClpB-K476C. (F and G) ATPase activities of ClpB-K476C/ClpB-K476C/E279A/E678A (F) and MDH disaggregation of ClpB-WT/ClpB-E279A/E678A (G) mixes were determined (red, blue). They are compared with curves calculated from a model (black to gray) that assumes that a mixed hexamer only displays ATPase or disaggregation activity if it contains the number of wild-type subunits indicated. Mixing ratios are indicated as number of E279A/E678A mutant subunits.

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Activation Assay, Activity Assay, Concentration Assay, Derivative Assay, Mutagenesis

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: Overview of Substrate-Bound ClpB-DWB-K476C (A) Left, top view, and middle and right, side views of the cryo-EM density map of the most populated conformation of casein-bound ClpB-DWB-K476C (KC-2). The six protomers form a closed ring with a helical arrangement of two stacked AAA tiers and a seam between subunits A and F. The flexible N-terminal domains, located above the AAA1 tier, are not visible at high contour level. M-domains are partly visible for protomers C–E. (B) Views of the cryo-EM maps of the three states of substrate-bound ClpB-DWB-K476C. Densities of protomers A and B are removed to show conformational changes in protomers AAA1E and AAA2F, highlighted by orange and red arrows, respectively. Orange and red hexagons show the position of moving AAA1E and AAA2F pore loops.

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Cryo-EM Sample Prep

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: Pore Loop Movements and Arginine Finger Contacts in the Three States of Substrate-Bound ClpB-DWB-K476C Suggest a Sequential Mechanism of ATP Hydrolysis and Substrate Threading (A) Pore loop interactions with the substrate in AAA1 (top panels) and AAA2 (bottom panels) rings. The pore loop AAA1E (orange) engages the substrate in KC-2, while the pore loop AAA2F (red) dissociates. AAA2F moves from the bottom to the top of the staircase of pore loops in KC-3. (B) Arginine finger engagements in the AAA1 and AAA2 ring. All protomers were aligned to the large lobe of AAA1 or AAA2 domain of protomer C to compare engagement of the arginine fingers with neighboring subunits. Arginine fingers of AAA1B–C and AAA2B–D are shown as gray ribbons and interact with the γ-phosphate of ATP bound at the active site of a neighboring subunit in all three states. Activity states of AAA2 protomers are indicated by green (active) and red (inactive) arrows. (C) Nucleotide densities for AAA2A, AAA2B, and AAA2F protomers and assigned nucleotide state.

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Activity Assay

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: Docked M-Domains Repress the Activity of ClpB-WT and Reduce the Range of AAA Domain Movements (A) Heterogeneity of M-domain conformations. Top and side views of the cryo-EM density maps for the two main M-domain conformations of the ClpB-DWB-K476C:casein complex and for the ClpB-DWB:casein complex. Detached M-domains are indicated by green arrows and docked M-domains by red arrows. (B) Docking states of M-domains: atomic models showing the predominant conformation of M-domains enclosing the AAA1 tier in ClpB-K476C (left) and ClpB-WT (right) states. In ClpB-WT, M-domains are docked in a horizontal conformation that is stabilized by head-to-tail interactions between motif 1 and motif 2 of neighboring M-domains. In ClpB-K476C, M-domains adopt a tilted conformation with motif1 contacting the AAA1 domain of the adjacent protomer. Head-to-tail interactions are broken, rendering M-domain motif 2 invisible in the cryo-EM maps. Here, full-length M-domains are shown, docked in the density of motif1, to emphasize the differences in M-domain docking states between ClpB-WT and ClpB-K476C.

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Activity Assay, Cryo-EM Sample Prep

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: Overview of Substrate-Bound ClpB-DWB, Pore Loop Movements and Arginine Finger Contacts (A) Views of the cryo-EM maps of the three states of substrate-bound ClpB-DWB. Densities of protomers A and B were removed to show conformational changes in protomers AAA1E and AAA2F, highlighted by orange and red arrows, respectively. Orange/red hexagons show the position of moving pore loops. (B) Interactions of ClpB-WT pore loops of the AAA1 (upper panel) and AAA2 (lower panel) rings with the substrate. The pore loop AAA2F (red) dissociates from the substrate in WT-2A. The pore loop AAA1E (orange) binds substrate in WT-2B. (C) Activity states of ClpB-WT AAA1 (upper panels) AAA2 (lower panels) domains. All protomers were aligned to the large AAA1 (AAA2) domain of protomer C to compare engagement of the arginine fingers with neighboring subunits. Arginine fingers of AAA1A-C and AAA2B-D are shown as grey ribbons and interact with the γ-phosphate of ATP bound at the active site of a neighboring subunit in all three states. Activity states of AAA1/2 protomers are indicated by green (active) and red (inactive) arrows. (D) Comparison of ClpB-WT and ClpB-K476C pore loop positions of AAA1E and AAA2F for states WT-1, WT-2A to WT-2B (pale to bright colors) and for states KC-1, KC-2 to KC-3 (pale to bright colors).

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Cryo-EM Sample Prep, Activity Assay

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet:

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Recombinant, ATPase Assay, Plasmid Preparation, Gel Extraction, Software, Electron Microscopy

Journal: Foods

Article Title: Recent Developments in Surface-Enhanced Raman Spectroscopy and Its Application in Food Analysis: Alcoholic Beverages as an Example

doi: 10.3390/foods11142165

Figure Lengend Snippet: Summary of the detection of harmful substances in alcoholic beverages using SERS in recent years.

Article Snippet: Sildenafil [ ] (white wine, wine, health wine, etc.) , Active substrate: Opto Trace Raman 202 (OTR 202) active colloids Instrument and parameter: (1) RmTracer-200-HS portable Raman spectrometer combined with a 785 nm excitation wavelength diode-stabilized stimulator (Opto Trace Technologies, Inc., Mountain View, CA, USA); (2) FEI Tecnai G2 F20 S-TWIN transmission electron microscope. a 785 nm excitation wavelength, a power of 200 mw, a scanning range of 200–3300 cm −1 , an optical resolution of 2 cm −1 , an integration time of 10 s, and an average spectral value of three times. , There was a good linear relationship between the intensity of Raman peak and the concentration of sildenafil in health wine and liquor. The Raman enhancement factor (EF) of OTR 202 colloids reached 1.84 × 10 7 and the limits of detection (LODs) of sildenafil in health wine and liquor were found to be as low as 0.1 mg/L. , (1) The Raman EF of OTR 202 colloids could reach 1.84 × 10 7 (2) The proposed method showed good performance.

Techniques: Concentration Assay, Raman Spectroscopy, Microscopy, Diffusion-based Assay, Activity Assay, Transmission Assay, Functional Assay, Modification, High Throughput Screening Assay, Imaging, Electron Microscopy