Marco Stenta

Electrostatic Control of the Photoisomerization Efficiency and Optical Properties in Visual Pigments: On the Role of Counterion Quenching

Hybrid QM(CASPT2//CASSCF/6-31G*)/MM(Amber) computations have been used to map the photoisomerization path of the retinal chromophore in Rhodopsin and explore the reasons behind the photoactivity efficiency and spectral control in the visual pigments. It is shown that while the electrostatic environment plays a central role in properly tuning the optical properties of the chromophore, it is also critical in biasing the ultrafast photochemical event: it controls the slope of the photoisomerization channel as well as the accessibility of the S(1)/S(0) crossing space triggering the ultrafast decay. The roles of the E113 counterion, the E181 residue, and the other amino acids of the protein pocket are explicitly analyzed: it appears that counterion quenching by the protein environment plays a key role in setting up the chromophores optical properties and its photochemical efficiency. A unified scenario is presented that discloses the relationship between spectroscopic and mechanistic properties in rhodopsins and allows us to draw a solid mechanism for spectral tuning in color vision pigments: a tunable counterion shielding appears as the elective mechanism for L<-->M spectral modulation, while a retinal conformational control must dictate S absorption. Finally, it is suggested that this model may contribute to shed new light into mutations-related vision deficiencies that opens innovative perspectives for experimental biomolecular investigations in this field.

Merging Organocatalysis with an Indium(III)‐Mediated Process: A Stereoselective α‐Alkylation of Aldehydes with Allylic Alcohols

Keywords: aldehydes ; allylic alcohols ; indium ; MacMillan catalysts ; organocatalysis ; Nucleophilic-Substitution ; 1 ; 3-Dicarbonyl Compounds ; Acids ; Benzylation ; Catalysis ; Aminocatalysis ; Activation ; Complexes ; Chemistry ; Spectra Reference EPFL-ARTICLE-150684doi:10.1002/chem.201001693View record in Web of Science Record created on 2010-08-31, modified on 2017-05-12

In situ structural analysis of the Yersinia enterocolitica injectisome

Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI: http://dx.doi.org/10.7554/eLife.00792.001

Adenine deactivation in DNA resolved at the CASPT2//CASSCF/AMBER level

We have employed hybrid CASPT2//CASSCF/AMBER calculations to map the (1)L(a)(1pipi*) deactivation path of a single quantum mechanical adenine in a d(A)(10).d(T)(10) double strand in water that is treated at the molecular mechanics level. We find that (a) the L(a) relaxation route is flatter in DNA than in vacuo and (b) the L(a) relaxation energy in DNA is much larger than the stabilization energy of the corresponding L(a) excimer. An intra-monomer relaxation process is found to be compatible with the multiexponential decay recorded in DNA, possibly including the longer (4100 ps) lifetime component.

Asymmetric Phase-Transfer-Catalyzed Intramolecular N-Alkylation of Indoles and Pyrroles: A Combined Experimental and Theoretical Investigation

Asymmetric phase-transfer catalysis (PTC) has risen to prominence over the last decade as a straightforward synthetic methodology for the preparation of pharmacologically active compounds in enantiomerically pure form. However, the complex interplay of weak nonbonded interactions (between catalyst and substrate) that could account for the stereoselection in these processes is still unclear, with tentative pictorial mechanistic representations usually proposed. Here we present a full account dealing with the enantioselective phase-transfer-catalyzed intramolecular aza-Michael reaction (IMAMR) of indolyl esters, as a valuable synthetic tool to obtain added-value compounds, such as dihydro-pyrazinoindolinones. A combined computational and experimental investigation has been carried out to elucidate the key mechanistic aspects of this process.

Molecular Simulations Highlight the Role of Metals in Catalysis and Inhibition of Type II Topoisomerase

Type II topoisomerase (topoII) is a metalloenzyme targeted by clinical antibiotics and anticancer agents. Here, we integrate existing structural data with molecular simulation and propose a model for the yet uncharacterized structure of the reactant state of topoII. This model describes a canonical two-metal-ion mechanism and suggests how the metals could rearrange at the catalytic pocket during enzymatic turnover, explaining also experimental evidence for topoII inhibition. These results call for further experimental validation.

Length control of the injectisome needle requires only one molecule of Yop secretion protein P (YscP)

The needle length of the Yersinia spp. injectisome is determined by Yop secretion protein P (YscP), an early substrate of the injectisome itself. There is a linear correlation between the length of YscP and the length of the needle, suggesting that YscP acts as a molecular ruler. However, it is not known whether one single molecule of YscP suffices to control the length of one needle or whether several molecules of YscP are exported in alternation with the needle subunit YscF until the needle length matches the ruler length, which would stop needle growth. To address this question, three different strains expressing simultaneously a short and a long version of YscP were engineered. The experimentally obtained needle length distribution was compared with the distributions predicted by stochastic modeling of the various possible scenarios. The experimental data are compatible with the single ruler model and not with the scenarios involving more than one ruler per needle.

The Lipopolysaccharide from Capnocytophaga canimorsus Reveals an Unexpected Role of the Core-Oligosaccharide in MD-2 Binding

Capnocytophaga canimorsus is a usual member of dogs mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a “hybrid backbone” lacking the 4′ phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 – lipid A complex in case the 4′ phosphate is not present.

Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis requires the ESX‐1 secretion system for full virulence. EspR plays a key role in ESX‐1 regulation via direct binding and transcriptional activation of the espACD operon. Here, we describe the crystal structures of EspR, a C‐terminally truncated form, EspRΔ10, as well as an EspR–DNA complex. EspR forms a dimer with each monomer containing an N‐terminal helix–turn–helix DNA binding motif and an atypical C‐terminal dimerization domain. Structural studies combined with footprinting experiments, atomic force microscopy and molecular dynamic simulations allow us to propose a model in which a dimer of EspR dimers is the minimal functional unit with two subunits binding two consecutive major grooves. The other two DNA binding domains are thus free to form higher‐order oligomers and to bridge distant DNA sites in a cooperative way. These features are reminiscent of nucleoid‐associated proteins and suggest a more general regulatory role for EspR than was previously suspected.

Deciphering Intrinsic Deactivation/Isomerization Routes in a Phytochrome Chromophore Model

High level ab initio correlated (CASPT2) computations have been used to elucidate the details of the photoinduced molecular motion and decay mechanisms of a realistic phytochrome chromophore model in vacuo and to explore the reasons underneath its photophysical/photochemical properties. Competitive deactivation routes emerge that unveil the primary photochemical event and the intrinsic photoisomerization ability of this system. The emerged in vacuo based static (i.e., nondynamical) reactivity model accounts for the formation of different excited state intermediates and suggests a qualitative rationale for the short (picosecond) excited state lifetime and ultrafast decay of the emission, its small quantum yield, and the multiexponential decay observed in both solvent and phytochromes. It is thus tentatively suggested that this is a more general deactivation scheme for photoexcited phytochrome chromophores that is independent of the surrounding environment. Spectroscopic properties have also been simulated in both isolated conditions and the protein that satisfactorily match experimental data. For this purpose, preliminary hybrid QM/MM computations at the correlated (CASPT2) level have been used in the protein and are reported here for the first time.