empty ribosomes  (New England Biolabs)

Journal: bioRxiv

Article Title: GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

doi: 10.1101/2024.08.12.607569

Figure Lengend Snippet: (A) Simplified functional cycle of GroEL/ES. (B) Stalled 70S ribosome:nascent chain complex (RNC). (C) Domain organisation and monomer structure (PDB: 6CVM) of E. coli β-galactosidase (β-gal). Positions of destabilising mutations I141N and G353D are shown in yellow. The first 510 residues of β-gal, corresponding to RNC 1-510 , are colored black. (D) GroEL binds preferentially to a conformationally destabilized RNC. Left: schematic of co-sedimentation assay. Right: Coomassie-stained SDS-PAGE of the resuspended ribosomal pellet. Prior to sedimentation, GroEL was incubated with either empty ribosomes (70S), wild-type RNC 1-510 (1-510 WT ), mutated (I141N, G353D) RNC 1-510mut (1-510 mut ), or RNC 1-1014 (1-1014 WT ). RNCs were purified from Δ tig cells. Bands corresponding to the NCs (*) and GroEL are indicated.

Article Snippet: For co-sedimentation assays, 1 μM RNCs or empty 70S ribosomes (NEB) were incubated (30 min, 30 °C) with (co-)chaperones (DnaK – 2-5 µM, TF – 2-5 µM, DnaJ – 1-2 µM, GroEL 14-mer – 2-5 μM, GroES 7-mer – 10-20 μM) in RNC low-salt buffer.

Techniques: Functional Assay, Sedimentation, Staining, SDS Page, Incubation, Purification

Journal: bioRxiv

Article Title: GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

doi: 10.1101/2024.08.12.607569

Figure Lengend Snippet: (A) Crosslinking-mass spectrometry (XL-MS) experiment. (B) GroEL crosslinks extensively to both the NC and ribosomal stalk proteins. Map of crosslinks between GroEL and RNC 1-510mut . Crosslinks between GroEL and the NC (orange) or L7/L12 (blue) are highlighted. (C) NCs crosslink to the inner surface of the GroEL cavity. Position of GroEL residues that crosslink to NC 1-510 , NC 1-510mut , or NC 1-1014 are shown in orange on the structure of GroEL (( Roh et al , 2017 ) PDB: 5W0S). Left, outer surface. Right, inner surface. (D) GroEL crosslinks to the L7/L12 stalk of empty ribosomes. Map of crosslinks between GroEL and empty 70S ribosomes. Crosslinks between GroEL L7/L12 (blue) are highlighted in blue. (E) L7/L12 crosslinks to the outer surface of GroEL. Position of GroEL residues that crosslink to L7/L12 in empty ribosomes, NC 1-510 , NC 1-510mut , or NC 1-1014 are shown in blue on the structure of GroEL (PDB: 5W0S). Left, outer surface. Right, inner surface.

Article Snippet: For co-sedimentation assays, 1 μM RNCs or empty 70S ribosomes (NEB) were incubated (30 min, 30 °C) with (co-)chaperones (DnaK – 2-5 µM, TF – 2-5 µM, DnaJ – 1-2 µM, GroEL 14-mer – 2-5 μM, GroES 7-mer – 10-20 μM) in RNC low-salt buffer.

Techniques: Mass Spectrometry

Journal: bioRxiv

Article Title: GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

doi: 10.1101/2024.08.12.607569

Figure Lengend Snippet: (A) Map of crosslinks between GroEL and RNC1-510WT. Crosslinks between GroEL and the NC (orange) or L7/L12 (blue) are highlighted. (B) Map of crosslinks between GroEL and RNC 1-1014 . Crosslinks between GroEL and the NC (orange) or L7/L12 (blue) are highlighted. (C) Map of crosslinks between GroEL and empty 70S ribosomes depleted of L7/L12. (D) Selective removal of L7/L12 from ribosomes. Top: Coomassie-stained SDS-PAGE of complete (70S) and L7/L12-depleted (70S-L7/12) ribosomes. The position of L7/12 is indicated by a black arrow. Bottom: Immunoblot of a replicate SDS-PAGE gel probed using antibodies against ribosomal proteins L7/12 and S2. (E) Removing L7/L12 from RNC 1-510mut does not prevent GroEL binding. Top: Coomassie-stained SDS-PAGE of the resuspended ribosomal pellet from a co-sedimentation assay. Prior to sedimentation, GroEL was incubated with either empty ribosomes (70S), 70S ribosomes after depletion of the ribosomal stalk (70S-L7/12), RNC 1-510mut (RNC 1-510 mut ), or RNC 1-510 mut after depletion of the ribosomal stalk (RNC 1-510 mut -L7/12). The pelleting assay for the last condition (RNC 1-510 mut -L7/12) was conducted in triplicate. Bands corresponding to the NCs (*) and GroEL are indicated. Bottom: Immunoblot of a replicate SDS-PAGE gel probed using antibodies against β-galactosidase and ribosomal proteins L7/12 and S2.

Article Snippet: For co-sedimentation assays, 1 μM RNCs or empty 70S ribosomes (NEB) were incubated (30 min, 30 °C) with (co-)chaperones (DnaK – 2-5 µM, TF – 2-5 µM, DnaJ – 1-2 µM, GroEL 14-mer – 2-5 μM, GroES 7-mer – 10-20 μM) in RNC low-salt buffer.

Techniques: Staining, SDS Page, Western Blot, Binding Assay, Sedimentation, Incubation

Journal: bioRxiv

Article Title: GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

doi: 10.1101/2024.08.12.607569

Figure Lengend Snippet: (A) Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) experiment. (B) Protection of GroEL induced by RNC binding. Difference in deuterium uptake after 10 s (grey) or 100 s (black), between GroEL bound to RNC 1-510mut and isolated GroEL. Values are plotted for individual GroEL peptides. Negative values indicate less deuteration of a peptide in RNC-bound GroEL relative to isolated GroEL, and sites with a difference in deuterium uptake > 0.5 Da are colored blue. (C) Sites of RNC-induced protection from deuterium exchange, mapped onto the domain organization and predicted structure of monomeric GroEL (AF-P0A6F5-F1). Crosslink sites for RNC1-510mut are indicated on the domain schematic with orange asterisks. (D) As in (C), shown on a top-view tetradecameric GroEL. The C-tails protrude into the central cavity. (E) As in (C), showing a side-view of tetradecameric GroEL (PDB: 5W0S). Left, outer surface. Right, inner surface. GroEL residues that crosslinked to NC 1-510mut are shown in orange. Sites with a difference in deuterium uptake > 0.5 Da are colored blue. (F) Visualization of GroEL:RNC assemblies. Left: Negative stain electron microscopy (nsEM) micrograph of DSBU-crosslinked GroEL:RNC 1-510mut assemblies. The scale bar corresponds to 100 nm. Examples of GroEL molecules positioned near ribosomes are circled (1-4). Right: 2D class averages of GroEL and 70S ribosomes. (G) NC density spans the GroEL cavity at the apical domains. 3D reconstruction of DSBU-crosslinked GroEL:RNC 1-510mut (map iv) from nsEM, viewed from the top and side. The structure of GroEL (( Roh et al , 2017 ) PDB: 5W0S) is docked into the map, and excess density corresponding to the NC is coloured black.

Article Snippet: For co-sedimentation assays, 1 μM RNCs or empty 70S ribosomes (NEB) were incubated (30 min, 30 °C) with (co-)chaperones (DnaK – 2-5 µM, TF – 2-5 µM, DnaJ – 1-2 µM, GroEL 14-mer – 2-5 μM, GroES 7-mer – 10-20 μM) in RNC low-salt buffer.

Techniques: Mass Spectrometry, Binding Assay, Isolation, Staining, Electron Microscopy

Journal: bioRxiv

Article Title: GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

doi: 10.1101/2024.08.12.607569

Figure Lengend Snippet: (A) Effect of different nucleotides on the stability of GroEL:RNC complexes. Coomassie-stained SDS-PAGE and immunoblot analysis of co-sedimentation assays of GroEL/ES with RNC 1-510mut incubated with different nucleotides. Both input (top) and pellet (bottom) fractions are shown. Prior to sedimentation, the RNC was incubated with GroEL either in low-salt RNC buffer (1), or with additional 1 mM ADP (2), ATP (3), ADP/BeF x (4) or ATP/BeF x (5). Alternatively, the RNC was incubated with GroEL/ES in low-salt RNC buffer (6) or with additional 1 mM ATP (7), ADP/BeF x (8) or ATP/BeF x (9). As controls, the RNC was incubated with a pre-formed complex of EL:ES 2 (10) in the presence of ATP/BeF x , or GroEL was incubated with empty 70S ribosomes (11). Any nucleotide and metal salts were present in the binding buffer as well as the sucrose cushion and wash buffers. Bands corresponding to the NCs (*) and GroEL are highlighted. Below each Coomassie-stained gel are immunoblots from the same gel probed using antibodies against GroES and ribosomal protein S2. (B) GroEL remains bound to RNCs upon addition of GroES and ATP/BeF x . Coomassie-stained SDS-PAGE of resuspended ribosomal pellets from co-sedimentation assays of GroEL with RNC 1-510mut . Where indicated, RNCs were incubated with GroEL, or GroEL, GroES and ATP/BeF x . As a control, GroEL, GroES and ATP/BeF x were pre-mixed to form symmetrically closed complexes before adding to RNCs (+[EL:ES] 2 ). Bands corresponding to the NCs (*) and GroEL are highlighted. Each lane corresponds to an independent co-sedimentation assay.

Article Snippet: For co-sedimentation assays, 1 μM RNCs or empty 70S ribosomes (NEB) were incubated (30 min, 30 °C) with (co-)chaperones (DnaK – 2-5 µM, TF – 2-5 µM, DnaJ – 1-2 µM, GroEL 14-mer – 2-5 μM, GroES 7-mer – 10-20 μM) in RNC low-salt buffer.

Techniques: Staining, SDS Page, Western Blot, Sedimentation, Incubation, Binding Assay, Control

Journal: bioRxiv

Article Title: GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

doi: 10.1101/2024.08.12.607569

Figure Lengend Snippet: (A) Map of crosslinks between GroEL/ES and RNC1-510mut. Crosslinks between GroEL and GroES (brown), from GroEL/ES to the NC (orange), and from GroEL/ES to L7/L12 (blue) are highlighted. (B) Crosslink sites are mapped onto the structures of the ATP/BeFx-stabilised EL:ES2 complex (PDB:7VWX), showing the outer surface. Residues are separated according to whether they crosslink to L7/L12 (blue), the NC (orange), or connect GroEL and GroES (brown). (C) Change in accessibility of GroEL residues upon GroES binding. Left: inner surface of the GroEL/ES cavity (left, PDB:7VWX). Right: inner (top) and outer (bottom) surfaces of apo-GroEL (PDB: 5W0S). Residues which crosslinked to the NC in the EL:ES 2 :RNC complex but not in the GroEL:RNC complex are shown in orange. (D) 2D class averages for double-capped GroEL (EL:ES2), single-capped GroEL (EL:ES1) and 70S ribosomes, from nsEM of the GroEL/ES:RNC complex. (E) Fourier Shell Correlation (FSC) plots for reconstructions obtained from nsEM analysis of EL:ES2:RNC complexes.

Article Snippet: For co-sedimentation assays, 1 μM RNCs or empty 70S ribosomes (NEB) were incubated (30 min, 30 °C) with (co-)chaperones (DnaK – 2-5 µM, TF – 2-5 µM, DnaJ – 1-2 µM, GroEL 14-mer – 2-5 μM, GroES 7-mer – 10-20 μM) in RNC low-salt buffer.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

doi: 10.1101/2024.08.12.607569

Figure Lengend Snippet: (A) TF and GroEL/ES do not compete for binding long NCs. Top: Coomassie-stained SDS-PAGE of the resuspended ribosomal pellet from co-sedimentation assays. Prior to sedimentation, RNC1-1014 was incubated with Trigger factor (+TF), GroEL with GroES and ATP/BeFx (+EL/ES), or a combination of the above in the specified order. Bands corresponding to the NC (*), TF and GroEL are indicated. Bottom: Immunoblot from an equivalent SDS-PAGE gel, probed against GroES and ribosomal protein S2. (B) DnaK and GroEL/ES compete for binding NCs. Top: Coomassie-stained SDS-PAGE of the resuspended ribosomal pellet from co-sedimentation assays. Prior to sedimentation, empty ribosomes (70S) or RNC1-510mut were incubated with GroEL (+EL), DnaK with DnaJ (+KJ), GroEL with GroES and ATP/BeFx (+EL/ES), or a combination of the above in the specified order. Bands corresponding to the NC (*), DnaK and GroEL are indicated. Bottom: Immunoblot from an equivalent SDS-PAGE gel, probed against GroES and ribosomal protein S2.

Article Snippet: For co-sedimentation assays, 1 μM RNCs or empty 70S ribosomes (NEB) were incubated (30 min, 30 °C) with (co-)chaperones (DnaK – 2-5 µM, TF – 2-5 µM, DnaJ – 1-2 µM, GroEL 14-mer – 2-5 μM, GroES 7-mer – 10-20 μM) in RNC low-salt buffer.

Techniques: Binding Assay, Staining, SDS Page, Sedimentation, Incubation, Western Blot