Bordetella pertussis Autotransporter Vag8 Binds Human C1 Esterase Inhibitor and Confers Serum Resistance

Bordetella pertussis employs numerous strategies to evade the immune system, including the ability to resist killing via complement. Previously we have shown that B. pertussis binds a complement regulatory protein, C1 esterase inhibitor (C1inh) to its surface in a Bvg-regulated manner (i.e. during its virulence phase), but the B. pertussis factor was not identified. Here we set out to identify the B. pertussis C1inh-binding factor. Using a serum overlay assay, we found that this factor migrates at approximately 100 kDa on an SDS-PAGE gel. To identify this factor, we isolated proteins of approximately 100 kDa from wild type strain BP338 and from BP347, an isogenic Bvg mutant that does not bind C1inh. Using mass spectrometry and bioinformatics, we identified the autotransporter protein Vag8 as the putative C1inh binding protein. To prove that Vag8 binds C1inh, vag8 was disrupted in two different B. pertussis strains, namely BP338 and 18–323, and the mutants were tested for their ability to bind C1inh in a surface-binding assay. Neither mutant strain was capable of binding C1inh, whereas a complemented strain successfully bound C1inh. In addition, the passenger domain of Vag8 was expressed and purified as a histidine-tagged fusion protein and tested for C1inh-binding in an ELISA assay. Whereas the purified Vag8 passenger bound C1inh, the passenger domain of BrkA (a related autotransporter protein) failed to do so. Finally, serum assays were conducted to compare wild type and vag8 mutants. We determined that vag8 mutants from both strains were more susceptible to killing compared to their isogenic wild type counterparts. In conclusion, we have discovered a novel role for the previously uncharacterized protein Vag8 in the immune evasion of B. pertussis. Vag8 binds C1inh to the surface of the bacterium and confers serum resistance

Modelling Time-Resolved Two-Dimensional Electronic Spectroscopy of the Primary Photoisomerization Event in Rhodopsin

Time-resolved two-dimensional (2D) electronic spectra (ES) tracking the evolution of the excited state manifolds of the retinal chromophore have been simulated along the photoisomerization pathway in bovine rhodopsin, using a state-of-the-art hybrid QM/MM approach based on multiconfigurational methods. Simulations of broadband 2D spectra provide a useful picture of the overall detectable 2D signals from the near-infrared (NW) to the near-ultraviolet (UV). Evolution of the stimulated emission (SE) and excited state absorption (ESA) 2D signals indicates that the S1 -> S-N (With N >= 2) ESAs feature a substantial blue-shift only after bond inversion and partial rotation along the cis -> trans isomerization angle, while the SE rapidly red-shifts during the photoinduced skeletal relaxation of the polyene chain. Different combinations of pulse frequencies are proposed in order to follow the evolution of specific ESA signals. These include a two-color 2DVis/NIR setup especially suited for tracking the evolution of the S-1 -> S-2 transitions that can be used to between different photochemical mechanisms of retinal photoisomerization as a function of the environment. The discriminate reported results are consistent with the available time-resolved pump-probe experimental data, and may be used for the design of more elaborate transient 2D electronic spectroscopy techniques

Modeling pH-dependent biomolecular photochemistry

International audienceThe tuning mechanism of pH can be extremely challenging to model computationally in complex biological systems, especially with respect to photochemical properties. This article reports a protocol aimed at modeling pH-dependent photodynamics, using a combination of constant-pH molecular dynamics and semi-classical nonadiabatic molecular dynamics simulations. With retinal photoisomerization in Anabaena Sensory Rhodopsin (ASR) as a testbed, we show that our protocol produces pH-dependent photochemical properties such as the isomerization quantum yield or decay rates. We decompose our results in single titrated residue contributions, identifying some key tuning amino acids. Additionally, we assess the validity of the single protonation state 1 picture to represent the system at a given pH and propose the most populated protein charge state as a compromise between cost and accuracy

Modeling pH-dependent biomolecular photochemistry

The tuning mechanism of pH can be extremely challenging to model computationally in complex biological systems, especially with respect to photochemical properties. This article reports a protocol aimed at modeling pH-dependent photodynamics, using a combination of constant-pH molecular dynamics and semi-classical nonadiabatic molecular dynamics simulations. With retinal photoisomerization in Anabaena Sensory Rhodopsin (ASR) as a testbed, we show that our protocol produces pH-dependent photochemical properties such as the isomerization quantum yield or decay rates. We decompose our results in single titrated residue contributions, identifying some key tuning amino acids. Additionally, we assess the validity of the single protonation state picture to represent the system at a given pH and propose the most populated protein charge state as a compromise between cost and accuracy

Probing the Photochemical Funnel of a Retinal Chromophore Model via Zero Point Energy Sampling Semiclassical Dynamics

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Relationship between Excited State Lifetime and Isomerization Quantum Yield in Animal Rhodopsins: Beyond the One-Dimensional Landau-Zener Model

We show that the speed of the chromophore photoisomerization of animal rhodopsins is not a relevant control knob for their light sensitivity. This result is at odds with the momentum-driven tunnelling rationale (i.e., assuming a one-dimensional Landau-Zener model for the decay: Zener, C. Non-Adiabatic Crossing of Energy Levels. Proc. R. Soc. London, Ser. A 1932, 137 (833), 696-702) holding that a faster nuclear motion through the conical intersection translates into a higher quantum yield and, thus, light sensitivity. Instead, a model based on the phase-matching of specific excited state vibrational modes should be considered. Using extensive semiclassical hybrid quantum mechanics/molecular mechanics trajectory computations to simulate the photoisomerization of three animal rhodopsin models (visual rhodopsin, squid rhodopsin and human melanopsin), we also demonstrate that phase-matching between three different modes (the reactive carbon and hydrogen twisting coordinates and the bond length alternation mode) is required to achieve high quantum yields. In fact, such "phase-matching" mechanism explains the computational results and provides a tool for the prediction of the photoisomerization outcome in retinal proteins

Chiral Pathways and Periodic Decay in <i>cis</i>-Azobenzene Photodynamics

Azobenzenes are candidates for efficient, photochemically triggered switching in devices of molecular size. The <i>cis</i>-azobenzene isomer is inherently chiral because of its helicity. Applying OM2/MRCI surface-hopping molecular dynamics simulations, we analyze chiral photoisomerization pathways in <i>cis</i>-azobenzene and correlate oscillatory features in the population decay to modes that trigger motion toward and from the S₁/S₀ crossing region