Journal: PLOS Biology

Article Title: The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains

doi: 10.1371/journal.pbio.3001995

Figure Lengend Snippet: ( A ) Bottom view (upper panel) and front view (lower panel) showing an overlay of the NatB-1 (pink) and NatB-2 (orange) ribosome structure with isolated densities for ribosome-bound NatA (bright green) (EMD-0201; ). ( B ) Comparison of positions for the NatA and NatB-2 catalytic subunits (Naa10 and Naa20) with respect to the 60S subunit shown as front and top view. The position of acetyl-CoA (Ac-CoA) and a putative model for the nascent chains is shown. For clarity, only Naa20 of NatB-2 is shown. ( C ) Left panel: cut front view of the NatB ribosome cryo-EM map highlighting the nascent polypeptide chain and the position NatB-1 and NatB-2 (left panel). Right panels: Zoom-in views highlighting the catalytic Naa20-2 subunit and illustration of the minimal distance that a nascent chain has to span to reach the active site of Naa20-2.

Article Snippet: A gene encoding a codon-optimized version of catalytically inactive E25A and H74A double mutant of Naa20 [ ] for E . coli expression was synthesized by Eurofins.

Techniques: Isolation, Comparison, Cryo-EM Sample Prep

Journal: Molecular Microbiology

Article Title: Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry

doi: 10.1111/mmi.12343

Figure Lengend Snippet: Comparison of phage and cellular RuvC resolving enzymes. A. Alignment of selected phage RuvC proteins. Structural elements of phage bIL67, Escherichia coli ( Eco ) and Thermus thermophilus ( Tth ) proteins are indicated above and below the aligned sequences (pink bars, α-helix; cyan arrows, β-sheet). Conserved residues are highlighted in red (acidic; active site), blue (basic), cyan (others) and orange (F69 in Ec RuvC and F73 in Tth RuvC). Every ten residues are labelled as filled grey circles above the bIL67 RuvC sequence. Filled magenta circles indicate the location of bIL67 RuvC S10A, T11A, K40A, R46A, S109A, K110A, K120A, R121A, R124A, K125A and R124A+K125A substitution mutants. Sequence conservation in E. coli and T. thermophilus RuvC is taken from the Pfam database entry, PF02075, highlighting the most highly conserved residues among bacterial RuvC family proteins. Conserved residues in phage RuvC are based on 24 orthologues using a similar approach. The percentage identity refers to homology of each protein with bIL67 RuvC. Green arrows indicate the positions of insertions to generate N- and C-terminal 67- Ec RuvC and Ec -67RuvC hybrids (see ). Accession numbers of selected lactococcal phage RuvC proteins from bIL67 (NP_042322), bIL66 (AAA99046), Q54 (YP_762587) and Streptococcus pyogenes inducible phage EJ-1 (NP_945263). B. Structural superimposition based upon their central β-sheets of monomers of 67RuvC (cyan) and Ec RuvC (dark magenta) shown in cartoon representation as in but with α-helices shown as cylinders. Helices and termini are labelled. C. Superimpostion of the catalytically critical residues at the active sites of 67RuvC (cyan), Ec RuvC (dark magenta) and Tth RuvC (olive green). Side-chains are shown in stick representation with the rest of the structure in cartoon form. The 67RuvC residue labelled D8 is an asparagine in the crystal structure shown. The Mg 2+ cation (green sphere) and solvent ligands (red spheres) from the 67RuvC structure are shown for reference. D. Alignment of dimers of 67RuvC and Ec RuvC based upon superimposition of the αB dimer interface helices. Structures are represented and coloured as in (B).

Article Snippet: An E. coli codon-optimized version ( ) of the Lactococcus lactis phage bIL67 ruvC ( ORF23 ) gene (Schouler et al ., ) was synthesized and inserted into the T7 expression vector pET24a(+) at NdeI and BamHI sites by GenScript to create p67RuvCwt.

Techniques: Comparison, Sequencing, Residue, Solvent

Journal: Nucleic Acids Research

Article Title: Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity

doi: 10.1093/nar/gkac565

Figure Lengend Snippet: Structural and dynamical analysis of PfAgo , TtAgo and CbAgo . ( A ) Thermal denature curves of p Ago s determined by nanoDSC, where the melting temperature ( T m ) values are presented. The solid arrows and dashed lines represent the onset of unfolding temperature ( T on ) and the physiological temperature ( T phy ), respectively. ( B ) Free-energy difference between a well-folded protein and a fully-unfolded state at 298 K derived from the urea-induced unfolding curve measured by far-UV CD spectra. The detailed procedure is presented in Materials and Methods and ref. . ( C ) Representation of the domain architectures of p Ago s. The PAZ domain, MID domain, N domain, and PIWI domain are colored in red, yellow, green, and blue, respectively. ( D ) Upper panel: Superposition of the crystalline structures of PfAgo (green, PDB ID: 1z26), TtAgo (blue, PDB ID: 4n47) and CbAgo (yellow, PDB ID: 6qzk). Lower panel: Schematic diagram of the domain organization of PfAgo , TtAgo and CbAgo . ( E ) The density of hydrogen bonds and salt bridges in PfAgo , TtAgo and CbAgo over the entire biomolecule obtained from MD simulation. ( F ) The number of hydrogen bonds and salt bridges formed between catalytic sites and surrounding amino acid residues in PfAgo , TtAgo and CbAgo obtained from MD simulation.

Article Snippet: A codon-optimized version of the PfAgo , TtAgo and CbAgo gene was synthesized by Sangon Biotech (Shanghai, China), and was cloned into the pET28a plasmid to construct pEX- PfAgo ( TtAgo and CbAgo ) with an N terminal His-tag.

Techniques: Derivative Assay, Circular Dichroism

Journal: Nucleic Acids Research

Article Title: Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity

doi: 10.1093/nar/gkac565

Figure Lengend Snippet: Structural analysis of tertiary and secondary structures in PfAgo , TtAgo and CbAgo at distinct temperatures obtained from far-UV CD spectroscopy and DSF spectroscopy. Far-UV CD spectra of ( A ) CbAgo , ( B ) TtAgo and ( C ) PfAgo measured at different temperatures. The insets in (A) and (B) are enlarged views showing intersecting portions of the far-UV CD spectra. The well-defined isochromatic point is only present in (A), indicating that the two-state scenario only works for the thermal unfolding process of CbAgo . Thermal unfolding curves of the three p Ago s measured by ( D ) far-UV CD spectroscopy and by ( E ) DSF spectroscopy. The solid arrows and dashed lines in (D) and (E) represent T on and T phy , respectively.

Article Snippet: A codon-optimized version of the PfAgo , TtAgo and CbAgo gene was synthesized by Sangon Biotech (Shanghai, China), and was cloned into the pET28a plasmid to construct pEX- PfAgo ( TtAgo and CbAgo ) with an N terminal His-tag.

Techniques: Circular Dichroism, Spectroscopy

Journal: Nucleic Acids Research

Article Title: Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity

doi: 10.1093/nar/gkac565

Figure Lengend Snippet: Structures and dynamics of PfAgo , TtAgo and CbAgo at moderate and elevated temperatures obtained from SAXS. Dimensionless Kratky plots of ( A ) CbAgo , ( B ) TtAgo and ( C ) PfAgo at different temperatures. The gray circles and green circles represent the SAXS data of globular protein (lysozyme, SASDBD ID: SASDMG2) and intrinsically disordered protein (SASDBD ID: SASDEE2), respectively for comparison. The atomic model and ab initio model of ( D ) CbAgo , ( E ) TtAgo and ( F ) PfAgo at 10°C and T phy . A comparison between the SAXS profile of proteins and the scattering profile obtained from the model is presented in . ( G ) R g of PfAgo , TtAgo and CbAgo at 10°C and T phy obtained from SAXS. The Guinier plots of proteins at different temperatures are shown in . Porod-Debye analysis, q 4 I ( q ), of ( H ) PfAgo and ( I ) TtAgo at 25°C and T phy . Transforming proteins at T phy by q 3 I ( q ) vs. q 3 (inset in Figure and ) verifies that the intensity decay is no longer q –4 but q –3 . The details about the SAXS data collection and analysis are summarized in .

Article Snippet: A codon-optimized version of the PfAgo , TtAgo and CbAgo gene was synthesized by Sangon Biotech (Shanghai, China), and was cloned into the pET28a plasmid to construct pEX- PfAgo ( TtAgo and CbAgo ) with an N terminal His-tag.

Techniques: Comparison

Journal: Nucleic Acids Research

Article Title: Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity

doi: 10.1093/nar/gkac565

Figure Lengend Snippet: The key role of the partially-disrupted structure of thermophilic p Ago s for cleavage of the ssDNA target at elevated temperatures. The thermal unfolding curves of ( A ) PfAgo and ( B ) TtAgo with and without incubation with urea as a function of temperature obtained from DSF. The arrow indicates the value of T on . The CK in (A) and (B) represents the mixture of urea and dye molecules without proteins, revealing that the observed temperature dependence of the fluorescence signals above is not caused by the interaction between the urea and dye molecules. Porod–Debye plots of ( C ) PfAgo and ( D ) TtAgo with and without urea. Cleavage activity of ( E ) PfAgo with and without 1.28 M urea and ( F ) TtAgo with and without 0.64 M urea, which was loaded with ssDNA guide and then incubated with ssDNA target in a 1:10:5 molar ratio (protein:guide:target). These activities are determined as the maximum slope of the time dependence of fluorescence intensity resulting from the cleavage function before it reaches the plateau (see ) . Three samples were used for each experimental condition. The results from three independent experiments were quantified. Error bars represent the standard deviations of three independent experiments. We note that the cleavage activity presented in ( E ) and ( F ) was obtained by using this fluorescence method instead of gel diagram analysis, as the former is more accurate . The detailed procedure is presented in the Materials and Methods. is another control experiment, revealing that urea by itself cannot degrade the target ssDNA. The nucleotide sequences of the guide and target ssDNAs are presented in . The cleavage activity of CbAgo incubated with 0.64 M urea at 37°C is shown in for comparison, such a low concentration of urea at such low temperature can affect neither the structure nor the activity of CbAgo .

Article Snippet: A codon-optimized version of the PfAgo , TtAgo and CbAgo gene was synthesized by Sangon Biotech (Shanghai, China), and was cloned into the pET28a plasmid to construct pEX- PfAgo ( TtAgo and CbAgo ) with an N terminal His-tag.

Techniques: Incubation, Fluorescence, Activity Assay, Control, Comparison, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Loosely-packed dynamical structures with partially-melted surface being the key for thermophilic argonaute proteins achieving high DNA-cleavage activity

doi: 10.1093/nar/gkac565

Figure Lengend Snippet: MD simulation results of PfAgo with and without 6 M urea at 37°C. ( A ) Superposition of the MD-derived structure of PfAgo with (blue) and without (red) the presence of urea. The simulation system does not include ssDNA guide, ssDNA target or Mn 2+ . The semitransparent circles highlight the regions where the significant changes in local structure occur when urea is added. The alignment on C alpha is achieved by using PyMOL software. The density of hydrogen bonds and salt bridges ( B ) globally and ( C ) around the catalytic sites in PfAgo with and without urea. Phase space sampled by MD on PfAgo ( D ) without and ( E ) with 6 M urea. RMSD is the root mean squared deviation of the structure in the first MD snapshot after equilibration. Comparison of B-factors of ( F ) the overall structure and ( G ) catalytic residues with and without urea. ( H ) Conformational deviations of the N domain in PfAgo (with or without urea) from that of the experimental functional form (i.e. the CbAgo -guide-target complex with Mg 2+ ), defined as the root mean squared deviations of C alpha . Here, the overall protein structure is aligned by PyMOL. ( I ) Structural alignment of PfAgo without (left panel) and with (right panel) urea with respect to that of the experimental functional conformation (PDB ID: 6qzk). The N domain in PfAgo without urea, with urea, and in the experimental functional form is highlighted in red, blue, and green, respectively.

Article Snippet: A codon-optimized version of the PfAgo , TtAgo and CbAgo gene was synthesized by Sangon Biotech (Shanghai, China), and was cloned into the pET28a plasmid to construct pEX- PfAgo ( TtAgo and CbAgo ) with an N terminal His-tag.

Techniques: Derivative Assay, Software, Comparison, Functional Assay