Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: Plots are the main chain amide chemical shift differences (Δδ for 1HN and 15N) between tSH2UN and either tSH2PM (A, C, and E) to examine the effect of IA negative charge, or tSH2FX (B, D, and F) to examine the effect of a flexible linker. Pairwise comparisons are made for the ligand‐free state (A and B), the ITP bound state (C and D), and the IHP‐bound state (E and F). For each pair of structures, n is the number of resonances resolved for both structures and shown in the plot. The distribution projected in each dimension is shown as a histogram along the top and right hand side (bin size: 0.01 ppm for Δδ1HN and 0.05 ppm for Δδ15N). Δδ values more than ~2σ from the mean of (A), that is the range [−0.05, 0.05] ppm for Δδ1HN or [−0.35, 0.35] ppm for Δδ15N, are colored red and labeled by residue; all other values are shown in blue. The Δδ values are mapped with the same colors onto the Syk tSH2 structure below each plot. The gray bars in E and F indicate that the SH2 domains are binding to the two isolated half IHP peptides instead of a full‐length ITP peptide. Only Δδ values corresponding to the (N)SH2 domain binding to C‐IHP and the (C)SH2 domain binding N‐IHP are plotted in E and F.

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques: Labeling, Binding Assay, Isolation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: The structure of Syk tandem SH2 (tSH2) (PDB ID 1A81, Chains A and B). PyMol session files for these representations are provided in Supplementary material. (A) tSH2 complex with a dp‐ITAM peptide (ITP, Ac‐PDpYEPIRKGQRDLpYSGLNQR‐NH2). Syk tSH2 is shown in ribbon representation. Green: (N)SH2 domain (8–118). Cyan: Interdomain A (IA, 119–162). Blue: (C)SH2 domain (163–264). Y130 is highlighted in red sticks. ITP is drawn with gold sticks, with the phosphoryl groups of the two phosphotyrosines shown as gold spheres to indicate the binding pockets. The interface between the three domains is illustrated by showing the side chains of residues that are within 6 Å of any heavy atom from either of the other two domains. (B) tSH2 with the domain‐domain interface highlighted using spheres for the interface residues defined as in A.

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: Schematic for Syk tSH2 interaction with dp‐ITAMs of membrane immunoreceptors. The two SH2 domains, (N)SH2 and (C)SH2, are labeled “N” and “C,” respectively, and connected by the IA linker. dp‐ITAM peptide binds in a head to tail orientation and is shown with each pYXX(I/L) cassette represented by an open square. (A) The 3‐state equilibrium binding model for Syk tSH2. The first step is intermolecular binding of the N‐terminal pYXX(I/L) cassette to (C)SH2. The second step is intra‐molecular binding, or isomerization, for the association of the C‐terminal pYXX(I/L) cassette to (N)SH2. (B–D) Models of different potential mechanisms for the increased binding free energy of the isomerization step (indicated by the asterisk) when Y130 in IA is phosphorylated (indicated by a circled “–” on IA to highlight the negative charge introduced by phosphorylation). (B) Suboptimal interactions in the (N)SH2 phosphotyrosine pocket: altered tSH2 domain–domain orientation from Y130 phosphorylation negatively affects binding interactions of the N(SH2) phosphotyrosine pocket, which lies at the domain interface. (C) Suboptimal dp‐ITAM conformation: altered tSH2 domain structure requires dp‐ITAM to adopt a suboptimal bound state conformation as a result of the change in the relative position of the two binding sites. (D) Entropy‐based model, in which Y130 phosphorylation increases domain–domain motion and thus entropy penalty for the isomerization step.

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques: Labeling, Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: DCOR values based on distance correlations of the chemical shift differences (Δδ) between free and bound states for the three forms of tSH2 in complex with ITP, N‐IHP and C‐IHP. Chemical shifts are from 84 residues with 15N HSQC assignments observed in all 12 tSH2 states. An IHP complex includes resonances of (N)SH2 residues for the C‐IHP/tSH2 complex and of (C)SH2 residues for the N‐IHP/tSH2 complex. The DCOR values were calculated either among the three IHP complexes only (black), or among these three IHP sets plus one of the ITP complexes (red for tSH2PM, blue dotted for tSH2FX, and green for tSH2UN).

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: Chemical shift differences (Δδ) for a given tSH2 protein binding to ITP with two pYXX(I/L) cassettes versus binding to IHP with one cassette. These differences are for binding with tSH2UN (A), tSH2PM (B), or tSH2FX (C). Color code and bin sizes are the same as Figure ​Figure5.5. Residues shown are the common set (n = 84) among all three proteins in all four states to minimize bias.

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques: Protein Binding, Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: The microscopic 3‐state binding process supported by NMR lineshape analysis is reconciled with the apparent 2 state binding from ITC data for the interactions between tSH2 constructs and ITP. (A) The microscopic 3 state process including thermodynamic parameters for intermolecular binding (K CN) and isomerization (KN′) required for global fitting of NMR titration data for multiple resonances from the two SH2 domains (Ref. 24). (B) The apparent 2 state binding relevant for interpreting the ITC titration curves.

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques: Binding Assay, Construct, Titration

Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: Apparent Thermodynamics Parameters Determined from ITC Data Fitted with a 2‐State Model a

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: Thermodynamics Parameters Calculated Using Eqs. (8) – (10) for the Isomerization Step in the Microscopic 3‐State Model of tSH2 Binding to ITP Peptide a

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Entropic allostery dominates the phosphorylation‐dependent regulation of Syk tyrosine kinase release from immunoreceptor tyrosine‐based activation motifs

doi: 10.1002/pro.3489

Figure Lengend Snippet: Departure from Thermodynamic Additivity for Bifunctional Binding of tSH2. Connection Thermodynamic Parameters for ITP Binding to the tSH2 Proteins from the Difference of the Equilibria for Isolated SH2 Domains Binding IHPs and tSH2 Proteins Binding ITP. a

Article Snippet: All plasmids are deposited in the non‐profit repository Addgene (Cambridge, MA) with the following plasmid#: 111269 (tSH2 UN ), 111271 (tSH2 PM ), 111272 (tSH2 FX ), 111274 (N‐SH2), and 111419 (C‐SH2).

Techniques: Binding Assay, Isolation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet:

Article Snippet: Plasmid: pET30a-Mpnd-RAMA , This study , Addgene plasmid 112226.

Techniques: Luciferase, Recombinant, SYBR Green Assay, Sequencing, Methylated DNA Immunoprecipitation, RNA Sequencing Assay, Plasmid Preparation, CRISPR, Software

Journal: eLife

Article Title: Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA

doi: 10.7554/eLife.95648

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pET-30a (plasmid) , Addgene , EMD Biosciences , Protein expression.

Techniques: Recombinant, Plasmid Preparation, Expressing, Chromatography, Protein Purification, Cloning, In Vitro, Amplification, Fluorescence, Sequencing, Software