Studying Weak and Dynamic Interactions of Posttranslationally Modified Proteins using Expressed Protein Ligation

Many cellular processes are regulated by posttranslational modifications that are recognized by specific domains in protein binding partners. These interactions are often weak, thus allowing a highly dynamic and combinatorial regulatory network of protein–protein interactions. We report an efficient strategy that overcomes challenges in structural analysis of such a weak transient interaction between the Tudor domain of the Survival of Motor Neuron (SMN) protein and symmetrically dimethylated arginine (sDMA). The posttranslational modification is chemically introduced and covalently linked to the effector module by a one-pot expressed protein ligation (EPL) procedure also enabling segmental incorporation of NMR-active isotopes for structural analysis. Covalent coupling of the two interacting moieties shifts the equilibrium to the bound state, and stoichiometric interactions are formed even for low affinity interactions. Our approach should enable the structural analysis of weak interactions by NMR or X-ray crystallography to better understand the role of posttranslational modifications in dynamic biological processes

Stereo overlay of the 20 lowest energy structures fitted to all residues except those in the closing base pair (U27–A43)

<p><b>Copyright information:</b></p><p>Taken from "The naturally occurring N6-threonyl adenine in anticodon loop of tRNA causes formation of a unique U-turn motif"</p><p>Nucleic Acids Research 2006;34(10):2878-2886.</p><p>Published online 31 May 2006</p><p>PMCID:PMC1474066.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p

Antibacterial activity of synthetic peptides in presence of apoplast fluid and in tomato fruits.

<p>(A) Approximately 10⁵ cfu/ml bacteria (<i>P. syringae</i> pv <i>tomato</i>) were incubated with 0 or 10 µg/ml peptide in the presence or absence of different concentrations (10 µg/ml or 30 µg/ml) of tomato apoplastic fluid. After 14–16 h the bacterial growth was determined by measuring OD_{600 nm}. APO, tomato apoplastic fluid. Values represent the mean of at least three biological replicates ± standard error of the mean. *indicates significantly different in comparison to the corresponding control treatment, <i>P</i><0.05. **indicates significantly different in comparison to the corresponding control treatment, <i>P</i><0.01. (B) <i>X. vesicatoria</i> (0.5×10⁵ cfu/ml) were treated with different concentrations of peptide SP10-5 and immediately injected into tomato fruits. After incubation for 5 d at room temperature infection symptoms were monitored. Above the values of incidence of infection symptoms is given in percentage. The total number of inoculation sides of three biological replicates were 22.</p

Growth inhibition of phytopathogens on tomato leaves.

<p>Tomato leaves were inoculated with (A) virulent <i>P. syringae pv. tomato</i> DC3000 (10⁷ CFU/ml) or (B) spores of <i>A. alternata</i> or <i>C. herbarum</i> (10⁴ spores/ml). Afterwards different concentrations of antimicrobial peptides were sprayed onto the leaves. Bacterial growth was monitored 30 min after peptide treatment by determining colony-forming units per defined leaf area. Fungal growth was analysed 48 h after peptide treatment by quantification of fungal DNA content in the leave tissue. Fungal growth on leaves treated with peptide dilution buffer was set to 100%. Values represent the mean of at least three biological replicates ± standard error of the mean. *indicates significantly lower than the control treatment, <i>P</i><0.05.</p

Sequences and structural-chemical properties of peptides of the 2^nd generation.

a<p>Estimated using the program Vector NTI 9.1 (Invitrogen).</p>b<p>Calculated using ProtParam tool (<a href="http://www.expasy.org/tools/protparam.html" target="_blank">http://www.expasy.org/tools/protparam.html</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071687#pone.0071687-Gasteiger1" target="_blank">[30]</a>), H [peptide], grand average hydrophobicity of full peptide.</p>c<p>H [cluster], hydrophobicity of the hydrophobic cluster of the peptides with the calculation based on the hydrophobicity scales for amino acids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071687#pone.0071687-Eisenberg1" target="_blank">[29]</a>. pI, isoelectric point. Special features: Important alterations in comparison to the leading structure are highlighted.>increased,</p

Effect of designed antimicrobial peptides on the viability of Arabidopsis mesophyll protoplasts <i>in-vitro</i>.

<p>Protoplasts were incubated for 1 h with different concentrations of SP1-1, SP10-2 and SP10-5 and analysed with a microscope (x 200). Cells with spherical shape without any sign of cytoplasmic degradation were defined as viable. A change of the cell shape, chloroplast release and/or agglomeration of protoplasts indicates a toxic effect of the peptides on plant cells (arrows).</p

Antimicrobial activities (MIC) and hemolytic activities of peptides of the 2^nd generation.

<p>Values reflect the MIC (µg/ml) after different incubation periods as indicated in brackets after the organism.</p>a<p>Shown are the peptide concentrations leading to 25% hemoglobin release from human blood cells.</p><p>>200 describes a slight hemolytic activity at 200 µg/ml but still below the above mentioned threshold, no value indicates none detectable hemolytic activity up to the highest concentration tested.</p

Bacterial membrane depolarization 60 minutes after AMP treatment.

<p>Depolarisation of bacterial membranes was determined by loading 5×10⁷ cfu/ml gram-positive <i>C. michiganensis</i> or gram-negative <i>P. syringae pv. syringae</i> with DiSC3(5) and measuring fluorescence intensity (FI) 60 min after addition of 0.5, 1, 5 or 10 µg/ml peptides (λ_ex: 622 nm λ_em: 670 nm). 1% Sodium dodecyl sulfate (SDS) and SP8 were used as positive and negative control, respectively. Shown are mean values ± standard error of the mean of three independent measurements normalized against FI after buffer treatment.</p

Antimicrobial activities (MIC) of designed first generation peptides against plant pathogens and hemolytic activities.

a<p>Shown are the peptide concentrations (µg/ml) leading to 25% hemoglobin release from human blood cells.</p><p>>200 describes a slight hemolytic activity at 200 µg/ml but still below the above mentioned threshold.</p

Sequences and structural-chemical properties of peptides of the 1^st generation.

a<p>Estimated using the program Vector NTI 9.1 (Invitrogen).</p>b<p>Calculated using ProtParam tool (<a href="http://www.expasy.org/tools/protparam.html" target="_blank">http://www.expasy.org/tools/protparam.html</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071687#pone.0071687-Gasteiger2" target="_blank">[84]</a>), H [peptide], grand average hydrophobicity of full peptide.</p>c<p>H [cluster], hydrophobicity of the hydrophobic cluster of the peptides with the calculation based on the hydrophobicity scales for amino acids (Eisenberg, 1984).</p>d<p>Secondary structure prediction according to NNPREDICT; H, helix; E, strand; -, no prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071687#pone.0071687-Kneller1" target="_blank">[31]</a>. pI, isoelectric point.</p