44 research outputs found
Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States
The bidirectional transformation
of a protein between its native
water-soluble and integral transmembrane conformations is demonstrated
for FraC, a hemolytic protein of the family of pore-forming toxins.
In the presence of biological membranes, the water-soluble conformation
of FraC undergoes a remarkable structural reorganization generating
cytolytic transmembrane nanopores conducive to cell death. So far,
the reverse transformation from the native transmembrane conformation
to the native water-soluble conformation has not been reported. We
describe the use of detergents with different physicochemical properties
to achieve the spontaneous conversion of transmembrane pores of FraC
back into the initial water-soluble state. Thermodynamic and kinetic
stability data suggest that specific detergents cause an asymmetric
change in the energy landscape of the protein, allowing the bidirectional
transformation of a membrane protein
MD simulations of Isd•heme•Isd ternary complexes.
<p>(A and D) Overall structures of the docked complexes IsdH-N3, IsdA-N, IsdC-N, and heme are represented in cyan, yellow, orange, and purple, respectively. The IsdH-N3•heme•IsdA-N and IsdA-N•heme•IsdC-N snapshots were obtained in MD simulations at 1,000 and 900 ns, respectively. (B and E) Plots of distances in MD trajectories; black, green, red, and blue traces represent distances between side-chain Oγ atoms of conserved serine residues and proximal carboxyl groups of heme, between Oη atoms of the primary tyrosine residues and iron, and between the secondary tyrosine and iron, respectively. (C and F) Plots of RMSD values; crystal structures were used as reference structures. Panel F: RMSD values that were calculated excluding the β7-β8 hairpin in IsdC-N (residues Asp-118 to Tyr-136) are presented in the blue trace.</p
Optimization of structures using the ONIOM method.
<p>Residues of IsdH-N3, IsdA-N, and IsdC-N are shown in cyan, yellow, and orange, respectively. Heme is shown in purple. (<b>A</b>) IsdH–Tyr642 and IsdA–Tyr170 are deprotonated in the IsdH-N3•heme•IsdA-N complex and (<b>B</b>) IsdA–Tyr166 and IsdC–Tyr132 are deprotonated in the IsdA-N•heme•IsdC-N complex.</p
Superposition of IsdH-N3 (cyan), IsdB-N2 (green), IsdA-N (yellow), and IsdC (orange) crystal structures.
<p>(A) Overview of Isd–NEAT domains; the heme molecule and primary/secondary tyrosine residues on the β8 strand are represented as ball-and-stick models; the iron atom of heme is shown as a green sphere. (B) Close-up view of the heme-binding pocket; heme propionate groups form H-bonds with conserved serine residues between the loop 1 region and the 3<sub>10</sub>-helix (IsdH–Ser563, IsdB–Ser361, IsdA–Ser82, or IsdC–Ser47).</p
Optimized structures with an additional proton.
<p>Residues of IsdH-N3, IsdA-N, and IsdC-N are shown in cyan, yellow, and orange, respectively. Heme is shown in purple. (A) Protonated IsdH–Tyr642 and–Tyr646; (B) protonated IsdA–Tyr166 and–Tyr170 in the IsdH-N3•heme•IsdA-N system; (C) protonated IsdA–Tyr166 and–Tyr170 in the IsdA-N•heme•IsdC-N system; (D) protonated IsdA–His83 and IsdC–Tyr136; an additional proton was initially placed near the Nδ atom of IsdA–His83.</p
Click Conjugation of a Binuclear Terbium(III) Complex for Real-Time Detection of Tyrosine Phosphorylation
Phosphorylation of proteins is closely
associated with various
diseases, and, therefore, its detection is vitally important in molecular
biology and drug discovery. Previously, we developed a binuclear TbÂ(III)
complex, which emits notable luminescence only in the presence of
phosphotyrosine. In this study, we conjugated a newly synthesized
binuclear TbÂ(III) complex to substrate peptides by using click chemistry.
Using these conjugates, we were able to detect tyrosine phosphorylation
in real time. These conjugates were superior to nonconjugated TbÂ(III)
complexes for the detection of tyrosine phosphorylation, especially
when the substrate peptides used were positively charged. Luminescence
intensity upon phosphorylation was enhanced 10-fold, making the luminescence
intensity of this system one of the largest among lanthanide luminescence-based
systems. We also determined Michaelis–Menten parameters for
the phosphorylation of various kinase/peptide combinations and quantitatively
analyzed the effects of mutations in the peptide substrates. Furthermore,
we successfully monitored the inhibition of enzymatic phosphorylation
by inhibitors in real time. Advantageously, this system detects only
the phosphorylation of tyrosine (phosphorylated serine and threonine
are virtually silent) and is applicable to versatile peptide substrates.
Our study thus demonstrates the applicability of this system for the
analysis of kinase activity, which could lead to drug discovery
MD simulations of Isd•heme•Isd ternary complexes.
<p>(A and D) Overall structures of the docked complexes IsdH-N3, IsdA-N, IsdC-N, and heme are represented in cyan, yellow, orange, and purple, respectively. The IsdH-N3•heme•IsdA-N and IsdA-N•heme•IsdC-N snapshots were obtained in MD simulations at 1,000 and 900 ns, respectively. (B and E) Plots of distances in MD trajectories; black, green, red, and blue traces represent distances between side-chain Oγ atoms of conserved serine residues and proximal carboxyl groups of heme, between Oη atoms of the primary tyrosine residues and iron, and between the secondary tyrosine and iron, respectively. (C and F) Plots of RMSD values; crystal structures were used as reference structures. Panel F: RMSD values that were calculated excluding the β7-β8 hairpin in IsdC-N (residues Asp-118 to Tyr-136) are presented in the blue trace.</p
Proposed heme transfer mechanism from IsdH-N3 to IsdC via IsdA.
<p>The sides of heme are shown in black and white. Heme is inverted upon formation of the complex between acceptor and donor NEAT domains.</p
Two-dimensional potential energy surface of the system of two phenols and an Fe(III)-porphine.
<p>Black contour lines are at intervals of 3 kcal/mol.</p
Supplementary data from Haemolytic actinoporins interact with carbohydrates using their lipid-binding module
Table S1. Structural homology of FraC; Figure S1. Kinetics of hemolysis by FraC in the presence of saccharides; Figure S2. Depiction of the lipid/carbohydrate binding region of FraC; Figure S3. Comparison of FraC with a fungal lectin