@article {2024|2169, title = {Structural profiles of the full phagocyte NADPH oxidase unveiled by combining computational biology and experimental knowledge}, journal = {J. Biol. Chem.}, volume = {300}, year = {2024}, pages = {107943}, abstract = {

The phagocyte NADPH oxidase (NOX2) is an enzyme, crucial for innate immune defense, producing reactive oxygen species necessary for pathogen destruction. Its activation requires the assembly of soluble proteins (p47phox, p40phox, p67phox, and Rac) with the membrane-bound flavocytochrome b558 (cytb558). We combined circular-dichroism analyses, with decades of experimental data, to filter structural models of the NADPH oxidase complex generated by the artificial intelligence program AlphaFold2 (AF2). The predicted patterns tend to closely resemble the active states of the proteins, as shown by the compact structure of the cytb558, whose dehydrogenase domain is stabilized closer to the membrane. The modeling of the interaction of p47phox with cytb558, which is the initial assembly and activation steps of the NADPH oxidase, enables us to describe how the C terminus of p47phox interacts with the cytb558. Combining the AF2 cytb558-p47phox model and its classical molecular dynamics simulations, we highlighted new hydrophobic lipid insertions of p47phox, particularly at residues Trp80-Phe81 of its PX domain. The AF2 models also revealed the implications of intrinsically disordered regions, such as the fragment between the PX domain and the SH3 regions of p47phox, in ensuring distant protein-protein and membrane-protein interactions. Finally, the AF2 prediction of the cytb558-Trimera model highlighted the importance of leaving Rac1 as a separate protein to reach an active state of the NADPH oxidase complex. Altogether, our step-by-step approach provides a structural model of the active complex showing how disordered regions and specific lipid and protein interactions can enable and stabilize the multisubunit assembly.

}, keywords = {alphafold, Circular Dichroism, Computational biology, lipid-protein interaction, membrane protein, molecular modeling, NADPH oxidase, NOX2, p47(phox), p67(phox), protein assembly, trimera}, author = {Aimeur, Sana and Fas, Burcu Aykac and Serfaty, Xavier and Santuz, Hubert and S Sacquin-Mora and Bizouarn, Tania and Taly, Antoine and Baciou, Laura} } @article {2020|2075, title = {Characterization of β-turns by electronic circular dichroism spectroscopy: a coupled molecular dynamics and time-dependent density functional theory computational study.}, journal = {Phys Chem Chem Phys}, volume = {22}, year = {2020}, month = {2020 Jan 21}, pages = {1611-1623}, abstract = {

Electronic circular dichroism is one of the most used spectroscopic techniques for peptide and protein structural characterization. However, while valuable experimental spectra exist for α-helix, β-sheet and random coil secondary structures, previous studies showed important discrepancies for β-turns, limiting their use as a reference for structural studies. In this paper, we simulated circular dichroism spectra for the best-characterized β-turns in peptides, namely types I, II, I\&$\#$39; and II\&$\#$39;. In particular, by combining classical molecular dynamics simulations and state-of-the-art quantum time-dependent density functional theory (with the polarizable embedding multiscale model) computations, two common electronic circular dichroism patterns were found for couples of β-turn types (namely, type I/type II\&$\#$39; and type II/type I\&$\#$39;), at first for a minimal di-peptide model (Ace-Ala-Ala-NHMe), but also for all sequences tested with non-aromatic residues in the central positions. On the other hand, as expected, aromatic substitution causes important perturbations to the previously found ECD patterns. Finally, by applying suitable approximations, these patterns were subsequently rationalized based on the exciton chirality rule. All these results provide useful predictions and pave the way for a possible experimental characterization of β-turns based on circular dichroism spectroscopy.

}, keywords = {Circular Dichroism, Computational Chemistry, Computer Simulation, Molecular Dynamics Simulation, Protein Conformation, beta-Strand, Protein Structure, Tertiary}, issn = {1463-9084}, doi = {10.1039/c9cp05776e}, author = {Migliore, Mattia and Bonvicini, Andrea and Tognetti, Vincent and Guilhaudis, Laure and Marc Baaden and Oulyadi, Hassan and Joubert, Laurent and S{\'e}galas-Milazzo, Isabelle} } @article {2018|2129, title = {Probing the quality control mechanism of the twin-arginine translocase with folding variants of a -designed heme protein.}, journal = {J Biol Chem}, volume = {293}, year = {2018}, month = {2018 05 04}, pages = {6672-6681}, abstract = {

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Tat system to recognize and translocate -designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the trimethylamine--oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme -induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system\&$\#$39;s quality-control mechanism.

}, keywords = {Amino Acid Sequence, Bacterial Proteins, Circular Dichroism, Escherichia coli, Escherichia coli Proteins, Heme-Binding Proteins, Hemeproteins, Membrane Transport Proteins, Methylamines, Models, Molecular, Oxidoreductases, N-Demethylating, Periplasm, Protein Folding, Protein Sorting Signals, Protein Stability, Protein Transport, Proton Magnetic Resonance Spectroscopy, Substrate Specificity, Temperature}, issn = {1083-351X}, doi = {10.1074/jbc.RA117.000880}, author = {Sutherland, George A and Grayson, Katie J and Adams, Nathan B P and Mermans, Daphne M J and Jones, Alexander S and Robertson, Angus J and Auman, Dirk B and Brindley, Amanda A and Sterpone, Fabio and Tuffery, Pierre and Philippe Derreumaux and Dutton, P Leslie and Robinson, Colin and Hitchcock, Andrew and Hunter, C Neil} } @article {2014|1717, title = {Lipid concentration and molar ratio boundaries for the use of isotropic bicelles.}, journal = {Langmuir}, volume = {30}, number = {21}, year = {2014}, month = {jun}, pages = {6162{\textendash}6170}, publisher = {Department of Chemistry, Universit{\'e} du Qu{\'e}bec {\`a} Montr{\'e}al and Centre Qu{\'e}b{\'e}cois sur les Mat{\'e}riaux Fonctionnels , P.O. Box 8888, Downtown Station, Montreal, Canada H3C 3P8.}, abstract = {Bicelles are model membranes generally made of long-chain dimyristoylphosphatidylcholine (DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensively used in the study of membrane interactions and structure determination of membrane-associated peptides, since their composition and morphology mimic the widespread PC-rich natural eukaryotic membranes. At low DMPC/DHPC (q) molar ratios, fast-tumbling bicelles are formed in which the DMPC bilayer is stabilized by DHPC molecules in the high-curvature rim region. Experimental constraints imposed by techniques such as circular dichroism, dynamic light scattering, or microscopy may require the use of bicelles at high dilutions. Studies have shown that such conditions induce the formation of small aggregates and alter the lipid-to-detergent ratio of the bicelle assemblies. The objectives of this work were to determine the exact composition of those DMPC/DHPC isotropic bicelles and study the lipid miscibility. This was done using (31)P nuclear magnetic resonance (NMR) and exploring a wide range of lipid concentrations (2-400 mM) and q ratios (0.15-2). Our data demonstrate how dilution modifies the actual DMPC/DHPC molar ratio in the bicelles. Care must be taken for samples with a total lipid concentration <=250 mM and especially at q \~{} 1.5-2, since moderate dilutions could lead to the formation of large and slow-tumbling lipid structures that could hinder the use of solution NMR methods, circular dichroism or dynamic light scattering studies. Our results, supported by infrared spectroscopy and molecular dynamics simulations, also show that phospholipids in bicelles are largely segregated only when q > 1. Boundaries are presented within which control of the bicelles{\textquoteright} q ratio is possible. This work, thus, intends to guide the choice of q ratio and total phospholipid concentration when using isotropic bicelles.}, keywords = {chemistry, Circular Dichroism, Detergents, Dimyristoylphosphatidylcholine, Fourier Transform Infrared, Light, Lipid Bilayers, Magnetic Resonance Spectroscopy, Materials Testing, Micelles, Molecular Dynamics Simulation, Phospholipid Ethers, Phospholipids, Radiation, Scattering, Solutions, Spectroscopy, Temperature}, doi = {10.1021/la5004353}, author = {Beaugrand, Ma\"{\i}wenn and Arnold, Alexandre A. and J{\'e}r{\^o}me H{\'e}nin and Warschawski, Dror E. and Williamson, Philip T F. and Marcotte, Isabelle} }