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An extremely bright, truly monomeric RFP, mScarlet, is described that outperforms existing RFPs in diverse labeling applications, especially in FRET with ratiometric imaging. We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.

Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

A new member of the Phosphotriesterase-Like Lactonases (PLL) family from the hyperthermophilic archeon Sulfolobus islandicus (SisLac) has been characterized. SisLac is a native lactonase that exhibits a high promiscuous phosphotriesterase activity. SisLac thus represents a promising target for engineering studies, exhibiting both detoxification and bacterial quorum quenching abilities, including human pathogens such as Pseudomonas aeruginosa. Here, we describe the substrate specificity of SisLac, providing extensive kinetic studies performed with various phosphotriesters, esters, N-acyl-homoserine lactones (AHLs) and other lactones as substrates. Moreover, we solved the X-ray structure of SisLac and structural comparisons with the closely related SsoPox structure highlighted differences in the surface salt bridge network and the dimerization interface. SisLac and SsoPox being close homologues (91% sequence identity), we undertook a mutational study to decipher these structural differences and their putative consequences on the stability and the catalytic properties of these proteins. We show that SisLac is a very proficient lactonase against aroma lactones and AHLs as substrates. Hence, data herein emphasize the potential role of SisLac as quorum quenching agent in Sulfolobus. Moreover, despite the very high sequence homology with SsoPox, we highlight key epistatic substitutions that influence the enzyme stability and activity.

miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving as a genetically-encodable light-induced source of singlet oxygen ( 1 O 2 ). We have determined its 1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization using an integrated approach combining spectroscopic and structural methods. Our results provide a structural framework to explain the ability of miniSOG to produce 1 O 2 as a competition between oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway has been identified that is pivotal for the performance of miniSOG as 1 O 2 photosensitizer, namely the photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1 O 2 quantum yield by one order of magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich residues that are progressively photo-oxidized, further contributing to facilitate the production of 1 O 2 . Our results help reconcile the apparent poor level of 1 O 2 generation by miniSOG and its excellent performance in correlative light and electron microscopy experiments.

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A . victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Organophosphates (OPs) are neurotoxic compounds for which current methods of elimination are unsatisfactory; thus bio-remediation is considered as a promising alternative. Here we provide the structural and enzymatic characterization of the recently identified enzyme isolated from Pseudomonas pseudoalcaligenes dubbed OPHC2. OPHC2 belongs to the metallo-β-lactamase superfamily and exhibits an unusual thermal resistance and some OP degrading abilities. The X-ray structure of OPHC2 has been solved at 2.1 Å resolution. The enzyme is roughly globular exhibiting a αβ/βα topology typical of the metallo-β-lactamase superfamily. Several structural determinants, such as an extended dimerization surface and an intramolecular disulfide bridge, common features in thermostable enzymes, are consistent with its high Tm (97.8°C). Additionally, we provide the enzymatic characterization of OPHC2 against a wide range of OPs, esters and lactones. OPHC2 possesses a broad substrate activity spectrum, since it hydrolyzes various phosphotriesters, esters, and a lactone. Because of its organophosphorus hydrolase activity, and given its intrinsic thermostability, OPHC2 is an interesting candidate for the development of an OPs bio-decontaminant. Its X-ray structure shed light on its active site, and provides key information for the understanding of the substrate binding mode and catalysis.

[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.

Sheet-on-sheet (SOS) fixed-target chips are arguably the most versatile, cheapest and simplest sample-delivery method for ambient-temperature data acquisition using serial crystallography approaches at synchrotrons and X-ray free-electron lasers (XFELs). Their defining feature, the absence of any hard-patterned restrictions around crystals, is their strength as it removes limitations on crystal sizes or environments. However, it is also their weakness when it comes to limiting undesired effects on yet-to-be-irradiated crystals due to diffusing heat, radicals or gas originating from previous exposures. We explored whether SOS chips can be used for damage-free serial data collection on the new ID29 beamline at the ESRF-EBS, a fourth-generation synchrotron light source, as well as at the new Cristallina-MX station at SwissFEL. We collected serial data sets from microcrystals of the hemoprotein DtpAa, which was reported to have a highly radiation-sensitive iron–water bond length. The data sets differ in step size between exposures within and between lines of a serpentine-like data-acquisition scan. We observe no significant changes in the distance of the water ligand of the heme in the structures obtained from the ID29 SSX data. However, when compared with those collected at Cristallina-MX, the diffraction intensities collected at ID29 suggest global damage akin to Bragg termination occurring during the 90 µs exposure at ID29. Moreover, differences in the heme geometry and the proximal histidine–iron bond length point to local damage in all ID29 data sets regardless of the X-ray spacing. SFX data collected at Cristallina-MX show a phase transition of the DtpAa crystal lattice for X-ray step sizes of ≤20 µm. This phase transition might be caused by heating and/or hydrogen-gas-induced crystal dehydration. Vigilance remains required to safeguard against radiation damage at fourth-generation synchrotrons and XFELs.

Carrying out macromolecular crystallography (MX) experiments at cryogenic temperatures significantly slows the rate of global radiation damage, thus facilitating the solution of high-resolution crystal structures of macromolecules. However, cryo-MX experiments suffer from the early onset of so-called specific radiation damage that affects certain amino-acid residues and, in particular, the active sites of many proteins. Here, a series of MX experiments are described which suggest that specific and global radiation damage are much less decoupled at room temperature than they are at cryogenic temperatures. The results reported here demonstrate the interest in reviving the practice of collecting MX diffraction data at room temperature and allow structural biologists to favourably envisage the development of time-resolved MX experiments at synchrotron sources.

The newly upgraded SLS 2.0 as a 4th generation synchrotron delivers unprecedented brilliance, enabling exploration of biological structures at vastly improved timescales and throughput. Our upgraded Macromolecular crystallography (MX) beamlines aim to support cutting-edge experiments including high-throughput fragment screening (FFCS),1 automated data collection,2 room temperature3, time-resolved serial crystallography,4 and X-ray scattering tensor tomography (SAS-TT)5—all generating massive data volumes requiring efficient management solutions. We are experimenting with different implementations including a multi-tiered approach utilizing HW-accelerated edge servers,6 PSI’s high- performance computing infrastructure, and DECTRIS CLOUD capabilities. The data processing pipelines operate both locally and remotely to support ongoing pilot experiments that will provide valuable insights into the performance and usability of these pipelines during user operation. This presentation will deliver firsthand experience from our active beamline commissioning in the beginning of SLS 2.0, sharing emerging strategies for handling high data rates. We will present our data acquisition, data processing and data reduction strategies. In addition, we will discuss the benefits and the challenges of our new data management systems. By sharing our current journey implementing these systems, we aim to contribute valuable perspectives to the MX community navigating similar challenges with increasing data volumes.