Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the ...currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins.
LOV domains are widespread photosensory modules that have also found applications in fluorescence microscopy, optogenetics, and light-driven generation of reactive oxygen species. Many of these ...applications require stable proteins with altered spectra. Here, we report a flavin-based fluorescent protein CisFbFP derived from Chloroflexus islandicus LOV domain-containing protein. We show that CisFbFP is thermostable, and its absorption and fluorescence spectra are red-shifted for ∼6 nm, which has not been observed for other cysteine-substituted natural LOV domains. We also provide a crystallographic structure of CisFbFP at the resolution of 1.2 Å that reveals alterations in the active site due to replacement of conservative asparagine with a serine. Finally, we discuss the possible effects of presence of cis-proline in the Aβ-Bβ loop on the protein's structure and stability. The findings provide the basis for engineering and color tuning of LOV-based tools for molecular biology.
•Chloroflexus islandicus genome encodes a red-shifted LOV domain.•Homologous mutation results in red shift of fluorescence spectra in YtvA but not in CagFbFP.•High-resolution crystal structure reveals altered active site and additional water molecule.•Presence of cis-proline in the Aβ-Bβ loop changes its structure in CisFbFP.
A study was carried out to investigate the influence of the initial powder features on the microstructure, the phase composition, and on the mechanical properties of WC-12%Co samples produced by ...binder jetting and densified by sintering in vacuum at 1400 °C and by sinter-HIPing. The initial powder consisted of a mixture of fine-grained and coarse-grained WC-based particles, and W2C-based ones. The density of the printed samples was 97.4% and 99.3% after sintering and sinter-HIPing, respectively. Layer-oriented porosity was observed only in sintered samples. The microstructure of the samples consisted of a mixture of fine and coarse WC grains, which can be the result of the coalescence of grains from anomalous coarse grains in the powder particles. Vickers hardness and transverse rupture strength of sinter-HIPed samples are 1205 HV and 2257 MPa, respectively, which is coherent with the microstructural analysis and close to coarse-grained commercial products.
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•Coarse grains in powder are responsible for abnormal grain growth during sintering.•Residual organic binder leads to W2C carburization and graphitic phase formation.•Sinter-HIPing is required to remove layer-oriented porosity after sintering.•Coarse grains are detrimental to Vickers hardness and transverse rupture strength.•Suitability of binder jetting for commercial-grade production is confirmed.
Under anaerobic conditions, bacteria may utilize nitrates and nitrites as electron acceptors. Sensitivity to nitrous compounds is achieved via several mechanisms, some of which rely on sensor ...histidine kinases (HKs). The best studied nitrate- and nitrite-sensing HKs (NSHKs) are NarQ and NarX from Escherichia coli. Here, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information. In particular, we show that around 6000 different NSHK sequences forming several distinct clusters may now be found in genomic databases, comprising mostly the genes from Beta- and Gammaproteobacteria as well as from Bacteroidetes and Chloroflexi, including those from anaerobic ammonia oxidation (annamox) communities. We show that the architecture of NSHKs is mostly conserved, although proteins from Bacteroidetes lack the HAMP and GAF-like domains yet sometimes have PAS. We reconcile the variation of NSHK sequences with atomistic models and pinpoint the structural elements important for signal transduction from the sensor domain to the catalytic module over the transmembrane and cytoplasmic regions spanning more than 200 Å.
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•Two ECAP cycles were applied to Zn-2Ag obtaining an ultra-fine-grained structure.•Isotropic nanoscale mechanical properties were obtained after ECAP.•The addition of Ag as alloying ...element in Zn reduced bacterial attachment.•ECAP processed Zn-2Ag alloy showed excellent antibacterial effect for S. Aureus.•ECAP processed Zn-2Ag alloy displayed uniformly degraded surface and corrosion rate.
Actual polymeric wound closure devices are not optimal for load-bearing applications due to the low mechanical properties and the risk of inflammation and bacterial infection mainly produced by multifilament and braided configurations. Biodegradable metallic Zn alloys are promising materials candidates; however, mechanical performance, corrosion behaviour, and biological response should be controlled in order to inhibit the risk of inflammation and bacterial infection. To this end, a Zn-2Ag (2 wt% Ag) alloy was processed by ECAP to evaluate the concurrent combined effect of grain refinement and Ag alloying on biodegradation and antibacterial activity. Two ECAP cycles were successfully applied to a Zn-2Ag alloy obtaining a homogeneous ultra-fine-grained structure in which nanoindentation maps suggested isotropic mechanical properties. Lower UTS and YS with higher elongation was reported after ECAP with similar corrosion rates as before processing. ECAP processed samples showed a homogeneous Ag+ release below the minimum inhibitory concentration for S. Aureus and no antibacterial effect was observed by diffusion. As expected, the presence of Ag in Zn-Ag alloys reduced bacterial attachment. Nevertheless, ECAP processed Zn-2Ag provided an excellent antibacterial activity after 3 h probably caused by the uniformly degraded and thus, non– stable, surface observed after bacterial adhesion.
In this paper, laser powder-bed fusion (L-PBF) additive manufacturing (AM) with a high-temperature inductive platform preheating was used to fabricate intermetallic TiAl-alloy samples. The gas ...atomized (GA) and mechanically alloyed plasma spheroidized (MAPS) powders of the Ti-48Al-2Cr-2Nb (at. %) alloy were used as the feedstock material. The effects of L-PBF process parameters—platform preheating temperature—on the relative density, microstructure, phase composition, and mechanical properties of printed material were evaluated. Crack-free intermetallic samples with a high relative density of 99.9% were fabricated using 900 °C preheating temperature. Scanning electron microscopy and X-Ray diffraction analyses revealed a very fine microstructure consisting of lamellar α2/γ colonies, equiaxed γ grains, and retained β phase. Compressive tests showed superior properties of AM material as compared to the conventional TiAl-alloy. However, increased oxygen content was detected in MAPS powder compared to GA powder (~1.1 wt. % and ~0.1 wt. %, respectively), which resulted in lower compressive strength and strain, but higher microhardness compared to the samples produced from GA powder.
This study investigates the influence of different sinter-HIP temperatures and binder saturation levels on the microstructure and properties of WC–12Co cemented carbide, produced using binder ...jetting. The sinter-HIP process was performed at 1400 °C, 1460 °C, and 1500 °C and binder saturation levels of 60% and 75% were selected during printing. The binder saturation proved to affect the repeatability of the manufacturing process and the sturdiness of the green models. The increase of the sintering temperature from 1400 °C to 1460 °C is correlated with an increase in the density. Nonetheless, a further raise in temperature to 1500 °C leads to significant grain coarsening without clear advantages in terms of porosity reduction. Both the transverse rupture strength and Vickers hardness increase when the sinter-HIP temperature rises from 1400 °C to 1460 °C, where the typical results for traditionally manufactured WC–12Co are met, with a comparable grain size. The transverse rupture strength and Vickers hardness then decrease for samples treated at 1500 °C. Finally, potential issues in the manufacturing process are identified and correlated with the defects in the final components.
Protein-fragment complementation assays are used ubiquitously for probing protein-protein interactions. Most commonly, the reporter protein is split in two parts, which are then fused to the proteins ...of interest and can reassemble and provide a readout if the proteins of interest interact with each other. The currently known split fluorescent proteins either can be used only in aerobic conditions and assemble irreversibly, or require addition of exogenous chromophores, which complicates the design of experiments. In recent years, light-oxygen-voltage (LOV) domains of several photoreceptor proteins have been developed into flavin-based fluorescent proteins (FbFPs) that, under some circumstances, can outperform commonly used fluorescent proteins such as GFP. Here, we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing protein from
, can serve as a split fluorescent reporter. We use the available genetic and structural information to identify three loops between the conserved secondary structure elements, Aβ-Bβ, Eα-Fα, and Hβ-Iβ, that tolerate insertion of flexible poly-Gly/Ser segments and eventually splitting. We demonstrate that the designed split pairs, when fused to interacting proteins, are fluorescent
in
and human cells and have low background fluorescence. Our results enable probing protein-protein interactions in anaerobic conditions without using exogenous fluorophores and provide a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based fluorescent reporters and optogenetic tools.
Light-oxygen-voltage (LOV) domains are common photosensory modules that found many applications in fluorescence microscopy and optogenetics. Here, we show that the Chloroflexus aggregans LOV domain ...can bind different flavin species (lumichrome, LC; riboflavin, RF; flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD) during heterologous expression and that its physicochemical properties depend strongly on the nature of the bound flavin. We show that whereas the dissociation constants for different chromophores are similar, the melting temperature of the protein reconstituted with single flavin species varies from ~ 60 °C for LC to ~ 81 °C for FMN, and photobleaching half-times vary almost 100-fold. These observations serve as a caution for future studies of LOV domains in non-native conditions yet raise the possibility of fine-tuning various properties of LOV-based fluorescent probes and optogenetic tools by manipulating the chromophore composition.
Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in ...fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2 °C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family.
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