F420H2-oxidase (FprA) catalyses the four-electron reduction of O2 to 2H2O using the reduced form of F420 as electron donor. The hydrophobic O2-channel detected by Kr-derivatization and the concerted ...movement of a gating loop could contribute to prevent unwanted side-reaction between the catalytic intermediates and solvents, therefore preventing reactive oxygen species formation.
The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing ...structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.
The recently developed red fluorescent protein Keima exhibits the largest Stokes shift (180 nm) observed to date. Combining X-ray crystallography with (in crystallo) UV−visible absorption, ...fluorescence, and Raman spectroscopy, we have investigated molecular determinants of this peculiar property. The results demonstrate a pH-dependent “reverse chromophore protonation” triggered by the key residue Asp157 and which couples to cis/trans isomerization of the chromophore. These data provided guidelines to rationally design a useful Keima variant.
KillerRed (KR) is a red fluorescent protein recognized as an efficient genetically encoded photosensitizer. KR generates reactive oxygen species via a complex process of photoreactions, ending up in ...photobleaching, the mechanism of which remains obscure. In order to clarify these mechanisms, we focus on a single mutant V44A (A44-KR) exhibiting the solely green component of KR. We report on the laser-induced structural transformations of A44-KR at cryogenic temperature, which we have investigated by combining UV–vis fluorescence/absorption spectroscopy with X-ray crystallography. Like the well-known GFP, A44-KR possesses a mixture of protonated (A) absorbing at 397 and deprotonated (B) absorbing at 515 nm chromophores, which are stressed by intense prolonged violet and blue laser sources. Both illuminations directly drive the B-chromophores toward a bleached trans isomerized form. A-type chromophores are sensitive only to violet illumination and are phototransformed either into a deprotonated green fluorescent form by decarboxylation of E218 or into a bleached form with a disordered p-hydroxybenzylidene. In crystallo spectroscopy at cryo-temperature allowed the identification and dissection of an exhaustive scheme of intermediates and end-products resulting from the phototransformation of A44-KR. This constitutes a framework for understanding the photochemistry of the photosensitizer KillerRed.
Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we ...previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (
T
m
= 73.5 °C; Δ
T
m
> 23 °C). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 Å and 1.6 Å resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the αβα-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance (∼27 Å) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts.
Illustration of cooperative thermostabilization effects of the double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site.
NiFe hydrogenases catalyze the reversible splitting of H₂ into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel ...network. While most NiFe hydrogenases are inactivated by O₂, a small subgroup, including the membrane-bound NiFe hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O₂ to water. This O₂ tolerance relies on a special 4Fe3S cluster that is capable of releasing two electrons upon O₂ attack. Here, the O₂ accessibility of the MBH gas tunnel network has been probed experimentally using a “soak-and-freeze” derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O₂ molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O₂ concentrations used for MBH crystal derivatization. The examination of the O₂-derivatized data furthermore uncovered two unexpected structural alterations at the 4Fe3S cluster, which might be related to the O₂ tolerance of the enzyme.
The red fluorescent protein KillerRed, engineered from the hydrozoan chromoprotein anm2CP, has been reported to induce strong cytotoxicity through the chromophore assisted light inactivation (CALI) ...effect. Here, we present the X-ray structures of KillerRed in its native and bleached states. A long water-filled channel is revealed, connecting the methylene bridge of the chromophore to the solvent. This channel facilitates the transit of oxygen and of reactive oxygen species (ROS) formed by reaction with the excited chromophore. The functional roles of key mutations used to produce KillerRed are discussed, strong chromophore distortions in the bleached state are revealed, and mechanisms for ROS production and self protection are proposed. The presence of a partially mature, photo-resistant, green-emitting state is characterized, which accounts for enhanced CALI by “pre-bleached” KillerRed.
Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. ...Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters. Bacteria have developed several Fe-S assembly systems, such as the ISC, NIF, and SUF systems. Interestingly, in
(
), the causative agent of tuberculosis (TB), the SUF machinery is the primary Fe-S biogenesis system. This operon is essential for the viability of
under normal growth conditions, and the genes it contains are known to be vulnerable, revealing the
SUF system as an interesting target in the fight against tuberculosis. In the present study, two proteins of the
SUF system were characterized for the first time: Rv1464(
) and Rv1465(
). The results presented reveal how these two proteins work together and thus provide insights into Fe-S biogenesis/metabolism by this pathogen. Combining biochemistry and structural approaches, we showed that Rv1464 is a type II cysteine-desulfurase enzyme and that Rv1465 is a zinc-dependent protein interacting with Rv1464. Endowed with a sulfurtransferase activity, Rv1465 significantly enhances the cysteine-desulfurase activity of Rv1464 by transferring the sulfur atom from persulfide on Rv1464 to its conserved Cys40 residue. The zinc ion is important for the sulfur transfer reaction between SufS and SufU, and His354 in SufS plays an essential role in this reaction. Finally, we showed that
SufS-SufU is more resistant to oxidative stress than
SufS-SufE and that the presence of zinc in SufU is likely responsible for this improved resistance. This study on Rv1464 and Rv1465 will help guide the design of future anti-tuberculosis agents.
NiFe hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and ...inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these NiFe hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2‐tolerant membrane‐bound NiFe hydrogenase of Ralstonia eutropha (ReMBH), using krypton‐pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2‐tolerant and O2‐sensitive NiFe hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.
At the end of the tunnel: The active site in NiFe hydrogenases is buried deep within the protein and accessible to gas‐phase substrates through tunnels. The structure of the O2‐tolerant, membrane‐bound NiFe hydrogenase of Ralstonia eutropha has now been determined from krypton‐derivatized protein crystals. The positions of the krypton atoms and corresponding calculations provided information on the gas tunnel network within the enzyme.
Abstract
Objective The intracavitary irradiation of cystic tumors has been used as a therapeutic alternative modality in the management of cystic craniopharyngiomas. In the present study, we review ...our experience, considering the technical issues, outcomes, and complications associated with the use of stereotactic intracavitary irradiation (SICI) with colloidal rhenium-186 (186Re) for cystic craniopharyngioma.
Material and Methods The records of 33 patients with cystic craniopharyngiomas treated by SICI with colloidal 186Re were retrospectively reviewed. The median radiation dose to the cyst wall was of 408 Gy (range: 175 Gy to 500 Gy). All tumors were composed of a large cyst cavity, and 9 (27.3%) also had a solid component. The mean follow-up period was of 3.7 years.
Results After SICI, 31 (93.9%) patients showed radiological evidence of cyst regression, and, in 2 (6.1%), no response was observed. An improvement in the visual deficits was observed in 8 cases (24.2%), and an improvement in endocrinogical disturbances, in 2 cases (6.1%). We observed complications in 3 patients (9.1%): diabetes insupidus in 1 case (3%), aggravation of visual acuity in 1 case (3%), and severe headache after infusion of the colloid in 1 case (3%); and 1 patient (3%) died after meningitis.
Conclusion Stereotactic intracavitary irradiation with colloidal 186Re is a safe procedure, with satisfactory results in the present series, and should be considered, in the management of cystic craniopharyngiomas, the first-intention therapy or as an adjuvant to other therapeutical modalities, with acceptable morbidity and mortality rates.
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