Isothermal titration calorimetry results and the curving fitting between peptide ligands and Shank1 PDZ protein.

<p>(A) The titration of ligands <b>p1</b>, <b>p2</b> and <b>p3</b> to Shank1 PDZ protein (A1, A2 and A3 are for <b>p1</b>, <b>p2</b> and <b>p3</b> respectively). (A1) [<b>p1</b>] = 4 mM, [Shank1 PDZ] = 400 μM; (A2) [<b>p2</b>] = 326 μM, [Shank1 PDZ] = 43 μM; (A3) [<b>p3</b>] = 354 μM, [Shank1 PDZ] = 35 μM. For the binding pair <b>p1</b> ligand and Shank1 PDZ, the affinity was very low so that protein concentration was adjusted higher to get accuracy affinity data. (B)The titration of diluted <b>p2</b>, <b>p3</b> and dimeric peptide with changed linker length to Shank1 PDZ protein. (B1) [<b>p2</b>] = 194 μM, [Shank1 PDZ] = 0.018 μM; (B2) [<b>p3</b>] = 160 μM, [Shank1 PDZ] = 16 μM; (B3) [dimeric peptide BM(PEG)₃] = 600 μM, [Shank1 PDZ] = 53 μM.</p

<i>R</i>_<i>eq</i> results from surface plasmon resonance and the curving fitting of ligand p1, p2 and p3 to Shank1 PDZ protein (Figure A, B, and C for ligand p1, p2 and p3 respectively).

<p>Fitting followed the steady state affinity model using this equation: here <i>R</i>_<i>eq</i> referred to response on the sensorgrams in the steady state region of the curve, <i>C</i> was concentration of analyte, <i>R</i>_<i>max</i> was the theoretical binding capacity, <i>n</i> = 1 in 1:1 binding model, <i>K</i>_<i>a</i> was association constant, and <i>K</i>_<i>d</i> could be obtained through 1/ <i>K</i>_<i>a</i>.</p

Additional file 2 of Trade-off in genome turnover events leading to adaptive evolution of Microcystis aeruginosa species complex

Supplementary Material

Surface plasmon resonance demonstrated ligands p1, p2 and p3 binding to Shank1 PDZ protein immobilized on the sensor chip.

<p>(A, B and C are for <b>p1</b>, <b>p2</b> and <b>p3</b> respectively). (A) [<b>p1</b>] = 11–346 μM; (B) [<b>p2</b>] = 10–320 μM; (C) [<b>p3</b>] = 5–168 μM. Dash lines in (B) & (C) were kinetics fitting used 1:1 Langmuir binding model for dimer <b>p2</b> and trimer <b>p3</b>.</p

Native mass spectrometric analysis of Shank1 PDZ protein with peptide dendrimer in 4:1 mixture.

<p>(A) Deconvoluted native mass spectrum of Shank1 PDZ protein without ligands, 91 μM in 150 mM ammonium acetate. (B-D) Deconvoluted mass spectra of Shank1 PDZ protein and peptide ligands <b>p1</b>, <b>p2</b> and <b>p3</b> mixed at 4:1 ratio (B, C and D are for <b>p1</b>, <b>p2</b> and <b>p3</b> respectively). Similarly, only protein−peptide complexes with 1:1 stoichiometry were observed (indicated by the solid line arrows). [Shank1 PDZ] = 45 μM; [peptide] = 11 μM. The peak at 13757 (indicated by * in the spectra) is from an impurity protein that co-purified with Shank1 PDZ protein in this batch. The protein peaks at around 14900 is from the Shank PDZ protein with the first methionine removed and the peak at around 15076 is from the Shank PDZ protein with the first methionine; both peaks are present in the spectrum of protein itself, so they are not relevant to the protein−peptide complex.</p

Functional Assembly of Protein Fragments Induced by Spatial Confinement

<div><p>Natural proteins are often confined within their local microenvironments, such as three-dimensional confinement in organelles or two-dimensional confinement in lipid rafts on cytoplasmic membrane. Spatial confinement restricts proteins' entropic freedom, forces their lateral interaction, and induces new properties that the same proteins lack at the soluble state. So far, the phenomenon of environment-induced protein functional alteration still lacks a full illustration. We demonstrate here that engineered protein fragments, although being non-functional in solution, can be re-assembled within the nanometer space to give the full activity of the whole protein. Specific interaction between hexahistidine-tag (His-tag) and NiO surface immobilizes protein fragments on NiO nanoparticles to form a self-assembled protein "corona" on the particles inside the nanopores of mesoporous silica. Site-specific assembly forces a shoulder-by-shoulder orientation and promotes fragment−fragment interaction; this interaction together with spatial confinement of the mesopores results in functional re-assembly of the protein half fragments. To our surprise, a single half fragment of luciferase (non-catalytic in solution) exhibited luciferase activity when immobilized on NiO in the mesopores, in the absence of the complimentary half. This shows for the first time that spatial confinement can induce the folding of a half fragment, reconstitute the enzyme active site, and re-gain the catalytic capability of the whole protein. Our work thereby highlights the under-documented notion that aside from the chemical composition such as primary sequence, physical environment of a protein also determines its function.</p></div

Split fragments of luciferase.

<p>(A) Luciferase assay. (B) Split luciferase to give two complementary fragments N-Luc 2–416 and C-Luc 398–550, each his-tagged at N terminus and C terminus respectively (the structure is from PDB ID 2D1S) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122101#pone.0122101.ref039" target="_blank">39</a>]. (C) SDS-PAGE picture of N-Luc and C-Luc.</p

SBA-NiO, a mesoporous silica material decorated with NiO nanoparticles, as the scaffolding support for protein fragments to interact.

<p>(A) Schematic illustration of the interaction of protein half fragments induced by site-specific interaction of His-tags with NiO nanoparticles in the cavities of mesoporous SBA material. (B) TEM image of as synthesized SBA-15. (C) TEM image of SBA-NiO.</p

Fragments of Luciferase assembled on SBA-NiO to form functional luciferase.

<p>(A) The assembly N-Luc/C-Luc/SBA-NiO catalyzed luciferin conversion, whereas N-Luc + C-Luc in solution remained inactive. a, N-Luc + C-Luc in solution; b, N-Luc/C-Luc/SBA-NiO. (B) Luciferase fragments alone on SBA-NiO N-Luc/SBA-NiO and C-Luc/SBA-NiO can regenerate the enzymatic activity. a, SBA-NiO; b, BSA/SBA-NiO; c, N-Luc/SBA-NiO; d, C-Luc/SBA-NiO; e, N-Luc/C-Luc/SBA-NiO. (C) Luciferase activity of N-Luc/C-Luc/SBA-NiO correlated with the amount of immobilized N-Luc and C-Luc.</p